乙酸检测试剂盒 Acetic Acid (ACS Manual Format) Assay Kit 货号:K-ACET Megazyme试剂盒

乙酸检测试剂盒

英文名:Acetic Acid (ACS Manual Format) Assay Kit

货号:K-ACET

规格:53 assays per kit

市场价: 2968

A simple method for the rapid and reliable measurement of acetic acid/acetate in foods, beverages and other materials. Content:53 assays per kit

Manual format UV-method for the determination of Acetic Acid in foodstuffs, beverages and other materials


Principle:
                           (acetyl-CoA synthetase)
(1) Acetic acid + ATP + CoA → acetyl-CoA + AMP + pyrophosphate

                                                (citrate synthase)
(2) Acetyl-CoA + oxaloacetate + H2O → citrate + CoA

                (L-malate dehydrogenase)
(3) L-Malate + NAD+ ↔ oxaloacetate + NADH + H+

Kit size:                            53 assays
Method:                            Spectrophotometric at 340 nm
Reaction time:                  ~ 14 min
Detection limit:                 0.14 mg/L
Application examples: 
Wine, beer, fruit and fruit juices, soft drinks, vinegar, vegetables, 
pickles, dairy products (e.g. cheese), meat, fish, bread, bakery products 
(and baking agents), ketchup, soy sauce, mayonnaise, dressings, 
paper (and cardboard), tea, pharmaceuticals (e.g. infusion solutions), 
feed and other materials (e.g. biological cultures, samples, etc.) 
Method recognition:     
Methods based on this principle have been accepted by EN, ISO, 
ICUMSA, IFU and MEBAK

Advantages

  • No wasted ACS solution (stable suspension supplied)
     
  • PVP incorporated to prevent tannin inhibition
     
  • All reagents stable for > 2 years after preparation

  • Very competitive price (cost per test)
     
  • Mega-Calc™software tool is available from our website for hassle-free raw data processing

Q1. Is the acetic acid kit specific for acetate?

Ethyl acetate, butyrate and propionate may react more slowly than acetate. Free fatty acids are not measured.

Q2. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q3. Does the decolourising preparation remove some VA during the process?

No, however the sample preparation process can be tested by adding a known amount of acetic acid standard and assessing the recovery of this.

Q4. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q5. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample,  in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. 6. State the sample type and describe the sample preparation steps if applicable.

Q6. What are the major differences between the various acetic acid test kits?

Megazyme produces 4 acetic acid test kits: 
K-ACET: uses the traditional ACS reaction.  Manual format for use with spectrophotometers.
K-ACETAF: uses the traditional ACS reaction.  Automated format for use with auto-analysers.
K-ACETAF:  uses the more recently developed and more rapid acetate kinase reaction.  Automated format for use with auto-analysers.
K-ACETAK: uses the more recently developed and more rapid acetate kinase reaction. Automated format for use with auto-analysers.
K-ACETRM: uses the more recently developed and more rapid acetate kinase reaction.  Manual format for use with spectrophotometers.

Q7. Can acetic acid be measured in culture/fermentation media?

Acetic acid in liquid cell culture media/supernatants or fermentation samples can be determined without any sample treatment (except clarification by centrifugation or filtration) and appropriate dilution in distilled water. 

Q8. Which acetic acid kit is recommended for a 96-well microplate format?

Auto-analysers use ~ 0.315 mL reaction volumes and pathlengths between 4-8 mm which is similar to a standard 96-well microplate where a 0.315 mL reaction volume would give a pathlength of ~ 6-7 mm.  Therefore K-ACETAK or K-ACETAF can be used directly in a 96-well microplate format with minimal assay optimisation.
If preferred, K-ACET or K-ACETRM may also be easily converted for use in a 96-well microplate format. Basically, the assay volumes for the cuvette format must bereduced approximately 10-fold for use in a 96-well microplate. However, some assay optimisation may be required (e.g. increased enzyme concentration etc.) and unlike the cuvette which has a set pathlength of 1 cm, the pathlength in the microplate is dependent upon the volume of liquid in the well.  Therefore to enable the calculation of the amount of analyte in the samples from tests performed in the microplate format one of the following must be done:

  1. The easiest method is to use a microplate reader that has a pathlength conversion capability (i.e. the microplate reader can detect the pathlength of each well and convert the individual readings to a 1 cm pathlength).  This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm pathlength) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values.


Acetic Acid Kit Recommendation For Microplate Format:
Either K-ACETRM or K-ACETAK is recommended for use in a 96-well microplate format and the main advantages / disadvantages are described below:
K-ACETRM:
The assay volumes of this kit should be reduced by 10-fold for use in a 96-well microplate format (some assay optimisation may be required, e.g. increased enzyme concentration etc.).
The calculation of results is achieved as outlined above in either of points 1, 2 or 3. 

Q9. Is the K-ACET Assay Kit suitable for measurement using cell culture media samples?

Yes, assuming that the concentration of the analyte in the sample (after sample preparation) is above   the limit of detection for the kit.  It may be sufficient to use the sample directly in the assay after clarification by centrifugation / filtering followed by dilution (if required) in distilled water. 

Q10. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q11. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q12. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q13. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q14. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

Q15. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:<a href="http://www.megaz

Megazyme 乙酸检测试剂盒K-ACET操作视频

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维生素C[L-抗坏血酸]检测试剂盒 L-Ascorbic Acid (L-Ascorbate) 货号:K-ASCO Megazyme试剂盒

维生素C[L-抗坏血酸]检测试剂盒

英文名:L-Ascorbic Acid (L-Ascorbate)

货号:K-ASCO

规格:40 assays (manual) / 400 assays

市场价: 2438

分析物意义:  蔬菜水果中的天然成分或加工食品中的添加成分

Megazyme检测试剂盒优点:反应快、试剂稳定 

For the specific assay of L-ascorbic acid in beverages, meat, flour, dairy and vegetable products. Content:40 assays per kit

Colourimetric method for the determination of L-Ascorbic Acid 
in foodstuffs, feed, wine and other materials

Principle:
                        (5-methylphenazinium methosulphate)
(1) L-Ascorbic acid + R-H2 + MTT → dehydroascorbate +                                                                                         MTT-formazan + H+
                            (ascorbic acid oxidase)
(2) L-Ascorbic acid + ½O2 → dehydroascorbate + H2O

Kit size:                             40 assays (manual) / 400 (microplate)
                                          / 400 (auto-analyser)
Method:                             Spectrophotometric at 578 nm
Reaction time:                   ~ 8 min
Detection limit:                  0.175 mg/L
Application examples:
Wine, beer, fruit juices, soft drinks, jam, milk, dairy products
(e.g. cheese), dietetic foods, baby foods, processed meat, baking
additives, fruit and vegetables (e.g. tomato and potato),
pharmaceuticals, feed and other materials (e.g. biological cultures,
samples, etc.)
Method recognition:    
Methods based on this principle have been accepted by MEBAK

Advantages

  • Very competitive price (cost per test)
     
  • All reagents stable for > 6 months after preparation
     
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
     
  • Standard included
     
  • Suitable for manual, microplate and auto-analyser formats 

 

Q1. Is it possible to adapt the kit for use on various auto-analysers?

Yes.  If the assay format is not available for your auto-analyser you can supply the following parameters of your relevant auto-analyser to Megazyme and an appropriate assay format may be available:
Parameters:
Instrument make and model:
Light-path length:
Reagent addition volumes (e.g. R1 volume, R2 volume, R3 if applicable):
Suggested auto-analyser format for K-ASCO:
Prepare the reagents as described in the K-ASCO data booklet then prepare R1 and R2 as follows:
Preparation of R1: (90 assays)

Component

Volume

Bottle 1

5.6 mL

Bottle 4

0.225 mL

Distilled water

17.5 mL

Total volume

23.325 mL

Preparation of R2: (90 assays)

Component

Volume

Bottle 2

2.25 mL

Bottle 3

2.25 mL

Total volume

4.5 mL

EXAMPLE METHOD:
R1: 0.260 mL
Sample: ~ 0.005 mL
R2: 0.05 mL
Note: For accurate measurement of ascorbic acid each sample requires two measurements, one with AAO (bottle 4) present and one with AAO (bottle 4) absent.
In the auto-analyser format described above there is no option to read A1 as in the cuvette assay.  In this instance use an ascorbic acid calibration curve to calculate results.  Alternatively add 0.025 mL of bottle 2 and 3 separately to mimic the cuvette assay and allow the reading  A1 (this will require 3 reagent additions) OR it may be possible to add the MTT (bottle 2) with R1 as follows:
Preparation of R1: (90 assays) 

Component

Volume

Bottle 1

5.6 mL

Bottle 2

2.25 mL

Bottle 4

0.225 mL

Distilled water

17.5 mL

Total volume

25.575 mL

Preparation of R2: (90 assays)

Component

Volume

Bottle 2

2.25 mL

Bottle 3

2.25 mL

Total volume

4.5 mL

EXAMPLE METHOD:
R1: 0.285 mL
Sample: ~ 0.005 mL
R2: 0.025 mL 

Q2. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q3. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q4. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.

柠檬酸检测试剂盒 Citric Acid Assay Kit 货号:K-CITR Megazyme试剂盒

柠檬酸检测试剂盒

英文名:Citric Acid Assay Kit

货号:K-CITR

规格:72 assays (manual) / 720 assays

市场价: 3180

分析物意义: 常见食品组分和添加剂 

Megazyme检测试剂盒优点:适用于手工和自动分析仪进行检测。重组的柠檬酸裂解酶-20摄氏度的稳定性>6个月。试剂稳定 

A flexible and simple method for the rapid and reliable measurement of citric acid (citrate) in foods, beverages and other materials. Content: 72 assays per kit

UV-method for the determination of Citric Acid in foods,
beverages and other materials

Principle:
(citrate lyase)
(1) Citrate → oxaloacetate + acetate

(L-malate dehydrogenase)
(2) Oxaloacetate + NADH + H+ → L-malate + NAD+

(D-lactate dehydrogenase)
(3) Pyruvate + NADH + H+ → D-lactate + NAD+

Kit size: 72 assays (manual) / 720 (microplate)
/ 840 (auto-analyser)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 5 min
Detection limit: 0.921 mg/L
Application examples:
Grape juice, wine, beer, fruit juices, soft drinks, tea, dairy products
(e.g. cheese), meat, processed meat, vegetable and fruit products,
bakery products, paper, pharmaceuticals, cosmetics and other
materials (e.g. biological cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by MEBAK, OIV,
EU, ISO2963, AOAC and IFU22
(Note: If the enzyme oxaloacetate decarboxylase is present in the sample, some
of the oxaloacetate product is converted to pyruvate. Therefore, to ensure citric
acid is measured quantitatively, D-lactate dehydrogenase (D-LDH) is employed
to efficiently convert any pyruvate produced into D-lactate and NAD+).

Advantages

  • Reconstituted citrate lyase stable for 4 weeks at 4°C / 6 months at -20°C
  • Buffer / cofactor / enzyme tablets for efficient use of kit components
  • PVP incorporated to prevent tannin inhibition
  • Very competitive price (cost per test)
  • Mega-Calc™software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Extended cofactors stability
  • Suitable for manual, microplate and auto-analyser formats

Q1. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q2. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q3. Is the Citric Acid test kit (K-CITR) suitable for measurement using cell culture media samples?

Yes, assuming that the concentration of the analyte in the sample (after sample preparation) is above the limit of detection for the kit.  It may be sufficient to use the sample directly in the assay after clarification by centrifugation / filtering followed but dilution (if required) in distilled water. 

Q4. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample,  in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q5. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q6. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q7. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q8. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q9. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q10. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

Q11. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q12. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q13. Is it possible to add a larger volume then 2 μL of enzyme to the microplate assay? In some instances 2 μL can be difficult to pipette manually.

Yes, instead of adding 2 μL of enzyme suspension an alternative is to dilute the enzyme and add a larger volume to the microplate assay.

Dilute the assay buffer 10-fold with distilled water and use this as the diluent to dilute an aliquot of the enzyme suspension also by 10-fold. Instead of 2 μL, use 20 μL of the diluted enzyme in the microplate assay.

Q14. When using this kit for quantitative analysis what level of accuracy and repeatability can be expected?

The test kit is extremely accurate – at Megazyme the quality control criteria for accuracy and repeatability is to be within 2% of the expected value using pure analytes.

However, the level of accuracy is obviously analyst and sample dependent.

Q15. Must the minimum absorbance change for a sample always be at least 0.1?

No. The 0.1 change of absorbance is only a recommendation. The lowest acceptable change in absorbance can is dictated by the analyst and equipment (i.e. pipettes and spectrophotometer) and therefore can be can be determined by the user. With accurate pipetting, absorbance changes as low as 0.02 can be used accurately.
If a change in absorbance above 0.1 is required but cannot be achieved due to low concentrations of analyte in a sample, this can be overcome by using a larger sample volume in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results. 

 

Megazyme 溶解淀粉 操作视频

Megazyme 试剂盒样品前处理准备操作视频

Megazyme 柠檬酸检测试剂盒Citric Acid操作视频(K-CITR)

D-乳酸[D-乳酸盐]检测试剂盒[快速] D-Lactic Acid (D-Lactate) (Rapid) Assay Kit 货号:K-DATE Megazyme试剂盒

D-乳酸[D-乳酸盐]检测试剂盒[快速]

英文名:D-Lactic Acid (D-Lactate) (Rapid) Assay Kit

货号:K-DATE

规格:50 assays (manual) / 500 assays

市场价: 3392

分析物意义:水果、蔬菜产品的质量指标 

Megazyme检测试剂盒优点:反应快、试剂稳定 

The D-Lactic Acid (D-Lactate) (Rapid) test kit is suitable for the rapid, specific measurement and analysis of D-lactic acid in wine, beer, juice, milk, cheese, vinegar, meat and other food products.
Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
Suitable for manual, auto-analyser and microplate formats.

UV-method for the determination of D-Lactic Acid in foodstuffs,
beverages and other materials

Principle:
(D-lactate dehydrogenase)
(1) D-Lactic acid + NAD+ ↔ pyruvate + NADH + H+

(glutamate-pyruvate transaminase)
(2) Pyruvate + D-glutamate → D-alanine + 2-oxoglutarate

Kit size: 50 assays (manual) / 500 (microplate)
/ 450 (auto-analyser)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 5 min
Detection limit: 0.21 mg/L
Application examples:
Wine, soft drinks, milk, dairy products (e.g. cream, milk / whey powder,
cheese, condensed milk and yogurt), foods containing milk (e.g. dietetic
foods, bakery products, baby food, chocolate, sweets and ice-cream),
vinegar, fruit and vegetables, processed fruit and vegetables, meat
products, food additives, paper (and cardboard), cosmetics,
pharmaceuticals and other materials (e.g. biological cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by DIN, GOST,
IDF, EEC, EN, ISO, OIV, IFU, AIJN and MEBAK

Advantages

  • Very rapid reaction with most samples (~ 5 min)
  • Very competitive price (cost per test)
  • All reagents stable for > 2 years after preparation
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Extended cofactors stability
  • Suitable for manual, microplate and auto-analyser formats

Q1. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample,  in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q2. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and   therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q3. Can perchloric acid be used to deproteinise/clarify samples prior to analysis using the D-Lactic Acid Assay Kit (K-DATE)? If so, how should such an extraction be performed?

Yes.  Perchloric acid extraction can be used in conjunction with this kit, and should be performed as follows:
WARNING: If you have not worked with perchloric acid before, you must consult your safety officer for advice.  Also, depending on the nature of the samples, it may be possible to reduce the concentration of perchloric acid, to for example 0.3 M (i.e. in the case of plasma).  It is thus very important to determine if this is possible for each type of sample used, in order to reduce the risk from working with concentrated perchloric acid.

Liquid samples:

  1. Carefully add an equal volume of ice cold 3 M perchloric acid and homogenise / fully disperse the sample (as appropriate).
  2. After 15 min incubation on ice (or in a refrigerator), centrifuge at 3000 x g for 15 min at 4°C.
  3. Neutralise by the slow addition of 2 M KOH.
  4. Incubate on ice (or in a refrigerator) until the potassium perchlorate has settled out by gravity (approximately 10 min), and then simply remove some of the clear supernatant and use directly in the assay.


Solid samples:

  1. Accurately weigh approx. 5 g of homogenised sample into a beaker containing 20 mL of 1 M perchloric acid and very carefully homogenise with an Ultraturrax® (or equivalent) for 5 min.
  2. Carefully add approx. 40 mL of distilled water and neutralise using 2 M KOH (using pH test strips for example).  Quantitatively transfer the contents to a 100 mL volumetric flask and fill to the mark with distilled water. If a fat layer develops, make sure this is above the mark, and the aqueous layer is at the mark.Incubate in a refrigerator for approx. 20 min to allow separation of fat and precipitation of potassium perchlorate.
  3. Filter through Whatman No. 1 filter paper, discarding the first few mL of filtrate, and use directly in the assay.

Q4. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q5. Is the D-Lactic Acid (D-Lactate) (Rapid) Assay Kit (K-DATE) suitable for measurement using cell culture media samples?

Yes, assuming that the concentration of the analyte in the sample (after sample preparation) is above the limit of detection for the kit.  It may be sufficient to use the sample directly in the assay after clarification by centrifugation/filtering followed, by dilution (if required) in distilled water. 

Q6. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q7. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q8. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q9. I have some doubts about the appearance/quality of a kit component what should be done9

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q10. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q11. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q12. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q13. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

 

Megazyme 溶解淀粉 操作视频

Megazyme 试剂盒样品前处理准备操作视频

Megazyme D-乳酸检测试剂盒D-Lactic Acid 操作视频(K-DATE)

D/L-乳酸[D-/L-乳酸盐]检测试剂盒 D/L-Lactic Acid (D-/L-Lacate) Assay Kit 货号:K-DLATE Megazyme试剂盒

D/L-乳酸[D-/L-乳酸盐]检测试剂盒

英文名:D/L-Lactic Acid (D-/L-Lacate) Assay Kit

货号:K-DLATE

规格:100 assays (50 of each) per kit

市场价: 5936

The D-/L-Lactic Acid (D-/L-Lactate) (Rapid) test kit is used for the rapid and specific concurrent measurement and analysis of L-lactic acid (L-lactate) and D-lactic acid (D-lactate) in beverages, meat, dairy and food products.
Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.

UV-method for the determination of D-/L-Lactic Acid in
foodstuffs, beverages and other materials

Principle:
(D-lactate dehydrogenase)
(1) D-Lactic acid + NAD+ ↔ pyruvate + NADH + H+

(L-lactate dehydrogenase)
(2) L-Lactic acid + NAD+ ↔ pyruvate + NADH + H+

(glutamate-pyruvate transaminase)
(3) Pyruvate + D-glutamate → D-alanine + 2-oxoglutarate

Kit size: 50 assays of each
Method: Spectrophotometric at 340 nm
Reaction time: ~ 10 min (L-lactic acid) and ~ 5 min (D-lactic acid)
Detection limit: 0.21 mg/L
Application examples:
Wine, soft drinks, milk, dairy products, foods containing milk (e.g. dietetic
foods, bakery products, baby food, chocolate, sweets and ice-cream),
vinegar, fruit and vegetables, processed fruit and vegetables, meat
products, food additives, paper (and cardboard), cosmetics, pharmaceuticals
and other materials (e.g. biological cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by DIN, GOST,
IDF, EEC, EN, ISO, OIV, IFU, AIJN and MEBAK

Advantages

  • Rapid total analysis time (concurrent / flexible D and L-lactic acid reaction format)
  • D-lactate dehydrogenase reaction very rapid with most samples (~ 5 min)
  • Very competitive price (cost per test)
  • All reagents stable for > 2 years after preparation
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Extended cofactors stability

 

Q1. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and   therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q2. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q3. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample,  in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q4. Is the D-/L-Lactic Acid (D-/L-Lactate) (Rapid) Assay Kit (K-DLATE) suitable for measurement using cell culture media samples?

Yes, assuming that the concentration of the analyte in the sample (after sample preparation) is above the limit of detection for the kit.  It may be sufficient to use the sample directly in the assay after clarification by centrifugation / filtering followed by dilution (if required) in distilled water. 

Q5. Can perchloric acid be used to deproteinise / clarify samples prior to analysis using the D-/L-Lactic Acid Assay Kit (K-DLATE)? If so, how should such an extraction be performed?

Yes.  Perchloric acid extraction can be used in conjunction with this kit, and should be performed as follows:
WARNING: If you have not worked with perchloric acid before, you must consult your safety officer for advice.  Also, depending on the nature of the samples, it may be possible to reduce the concentration of perchloric acid, to for example 0.3 M (i.e. in the case of plasma).  It is thus very important to determine if this is possible for each type of sample used, in order to reduce the risk from working with concentrated perchloric acid.

Liquid samples:

  1. Carefully add an equal volume of ice cold 3 M perchloric acid and homogenise / fully disperse the sample (as appropriate).
  2. After 15 min incubation on ice (or in a refrigerator), centrifuge at 3000 x g for 15 min at 4°C.
  3. Neutralise by the slow addition of 2 M KOH.
  4. Incubate on ice (or in a refrigerator) until the potassium perchlorate has settled out by gravity (approximately 10 min), and then simply remove some of the clear supernatant and use directly in the assay.


Solid samples:

  1. Accurately weigh approx. 5 g of homogenised sample into a beaker containing 20 mL of 1 M perchloric acid and very carefully homogenise with an Ultraturrax® (or equivalent) for 5 min.
  2. Carefully add approx. 40 mL of distilled water and neutralise using 2 M KOH (using pH test strips for example).  Quantitatively transfer the contents to a 100 mL volumetric flask and fill to the mark with distilled water.  If a fat layer develops, make sure this is above the mark, and the aqueous layer is at the mark.
  3. Incubate in a refrigerator for approx. 20 min to allow separation of fat and precipitation of potassium perchlorate.
  4. Filter through Whatman No. 1 filter paper, discarding the first few mL of filtrate, and use directly in the assay.

Q6. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q7. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q8. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q9. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q10. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q11. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q12. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q13. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

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Megazyme D-L-乳酸快速测定试剂盒操作视频(K-DLATE)

Megazyme 溶解淀粉 操作视频

Megazyme 试剂盒样品前处理准备操作视频

D-苹果酸检测试剂盒 D-Malic Acid Assay Kit 货号:K-DMAL Megazyme试剂盒

D-苹果酸检测试剂盒

英文名:D-Malic Acid Assay Kit

货号:K-DMAL

规格:100 assays (manual) / 1000 assays (micropl

市场价: 3700

The D-Malic Acid assay kit is suitable for the specific measurement and analysis of D-malic acid (D-malate) in beverages and food products.
Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
Suitable for manual, auto-analyser and microplate formats.

UV-method for the determination of D-Malic Acid in foodstuffs,
beverages and other materials

Principle:
(D-malate dehydrogenase)
(1) D-Malic acid + NAD+ → pyruvate + CO2 + NADH + H+

Kit size: 100 assays (manual) / 1000 (microplate)
/ 1100 (auto-analyser)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 6 min
Detection limit: 0.26 mg/L
Application examples:
Wine, beer, fruit juices, soft drinks, dietetic foods, candies, fruit and
vegetables, cosmetics, pharmaceuticals and other materials
(e.g. biological cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by EEC, EN,
DIN, OIV, IFU, and AIJN

Advantages

  • No wasted D-malate dehydrogenase solution (stable suspension supplied)
  • Very competitive price (cost per test)
  • All reagents stable for > 2 years after preparation
  • Rapid reaction (even with difficult samples)
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Extended cofactors stability
  • Suitable for manual, microplate and auto-analyser formats

Q1. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and   therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q2. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q3. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample,  in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q4. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q5. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q6. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q7. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q8. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q9. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

Q10. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q11. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q12. When using this kit for quantitative analysis what level of accuracy and repeatability can be expected?

The test kit is extremely accurate – at Megazyme the quality control criteria for accuracy and repeatability is to be within 2% of the expected value using pure analytes.

However, the level of accuracy is obviously analyst and sample dependent.

Q13. Is it possible to add a larger volume then 2 μL of enzyme to the microplate assay? In some instances 2 μL can be difficult to pipette manually.

Yes, instead of adding 2 μL of enzyme suspension an alternative is to dilute the enzyme and add a larger volume to the microplate assay.

Dilute the assay buffer 10-fold with distilled water and use this as the diluent to dilute an aliquot of the enzyme suspension also by 10-fold. Instead of 2 μL, use 20 μL of the diluted enzyme in the microplate assay.

Q14. Must the minimum absorbance change for a sample always be at least 0.1?

No. The 0.1 change of absorbance is only a recommendation. The lowest acceptable change in absorbance can is dictated by the analyst and equipment (i.e. pipettes and spectrophotometer) and therefore can be can be determined by the user. With accurate pipetting, absorbance changes as low as 0.02 can be used accurately.
If a change in absorbance above 0.1 is required but cannot be achieved due to low concentrations of analyte in a sample, this can be overcome by using a larger sample volume in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results. 

Q15. Can the sensitivity of the kit assay be increased?

Yes. Samples with the lower concentrations of analyte will generate a lower absorbance change. For samples with low concentrations of analyte, a larger sample volume can be used in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results.

Megazyme D-苹果酸检测试剂盒操作视频(K-DMAL)

Megazyme 溶解淀粉 操作视频

Megazyme 试剂盒样品前处理准备操作视频

甲酸检测试剂盒 Formic Acid Assay Kit 货号:K-FORM Megazyme试剂盒

甲酸检测试剂盒

英文名:Formic Acid Assay Kit

货号:K-FORM

规格:25 assays (manual) /250 assays

市场价: 2968

A simple method for the rapid and reliable measurement of formic acid (formate) in foods, beverages and other materials. Content:25 assays per kit

UV-method for the determination of Formic Acid in foods,
beverages and other materials

Principle:
(formate dehydrogenase)
(1) Formic acid + NAD+ → CO2 + NADH + H+

Kit size: 25 assays (manual) / 250 (microplate)
/ 220 (auto-analyser)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 12 min
Detection limit: 0.0932 mg/L
Application examples:
Wine, fruit juices, pickles, vinegar, jam, bakery products, honey, fish,
meat and other materials (e.g. biological cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by MEBAK

Advantages

  • No wasted formate dehydrogenase solution (stable suspension supplied)
  • Pyrazole incorporated to prevent alcohol dehydrogenase interference
  • Very competitive price (cost per test)
  • All reagents stable for > 2 years after preparation
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats

 

Q1. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample,  in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q2. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q3. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q4. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q5. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q6. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q7. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q8. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

Q9. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q10. When using this kit for quantitative analysis what level of accuracy and repeatability can be expected?

The test kit is extremely accurate – at Megazyme the quality control criteria for accuracy and repeatability is to be within 2% of the expected value using pure analytes.

However, the level of accuracy is obviously analyst and sample dependent.

Q11. Is it possible to add a larger volume then 2 μL of enzyme to the microplate assay? In some instances 2 μL can be difficult to pipette manually.

Yes, instead of adding 2 μL of enzyme suspension an alternative is to dilute the enzyme and add a larger volume to the microplate assay.

Dilute the assay buffer 10-fold with distilled water and use this as the diluent to dilute an aliquot of the enzyme suspension also by 10-fold. Instead of 2 μL, use 20 μL of the diluted enzyme in the microplate assay.

Q12. Must the minimum absorbance change for a sample always be at least 0.1?

No. The 0.1 change of absorbance is only a recommendation. The lowest acceptable change in absorbance can is dictated by the analyst and equipment (i.e. pipettes and spectrophotometer) and therefore can be can be determined by the user. With accurate pipetting, absorbance changes as low as 0.02 can be used accurately.
If a change in absorbance above 0.1 is required but cannot be achieved due to low concentrations of analyte in a sample, this can be overcome by using a larger sample volume in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results. 

Q13. Can the sensitivity of the kit assay be increased?

Yes. Samples with the lower concentrations of analyte will generate a lower absorbance change. For samples with low concentrations of analyte, a larger sample volume can be used in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results.

Megazyme 甲酸检测试剂盒操作视频(K-FORM)

Megazyme 溶解淀粉 操作视频

Megazyme 试剂盒样品前处理准备操作视频

D-3羟丁酸检测试剂盒 D-3-Hydroxybutyric Acid 货号:K-HDBA Megazyme试剂盒

D-3羟丁酸检测试剂盒

英文名:D-3-Hydroxybutyric Acid

货号:K-HDBA

规格:60 assays (manual) /

市场价: 3180

The D-3-Hydroxybutyric Acid Assay Kit is suitable for the specific measurement and analysis of D-hydroxybutyric acid in eggs and egg products and other foods and beverages. Suitable for manual, auto-analyser and microplate formats.

For the rapid and specific assay of D-lactic acid in wine, beer, juice, milk, cheese, vinegar and meat products. Content:50 assays per kit

 

Advantages

  • Very competitive price (cost per test)
  • All reagents stable for > 2 years after preparation
  • Very rapid reaction (~ 3 min)
  • No wasted diaphorase solution (stable suspension supplied)
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats

Colourimetric method for the determination of D-3-Hydroxybutyric Acid in foodstuffs

Principle:
(3-hydroxybutyrate dehydrogenase)
(1) D-3-Hydroxybutyrate + NAD+ ↔ acetoacetate + NADH + H+

(diaphorase)
(2) INT + NADH + H+ → NAD+ + INT-formazan

Kit size: 60 assays (manual) / 600 (microplate)
/ 740 (auto-analyser)
Method: Spectrophotometric at 492 nm
Reaction time: ~ 3 min
Detection limit: 0.20 mg/L
Application examples:
Egg, egg products (e.g. egg powder) and other materials (e.g. biological
cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by CEC

Q1. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q2. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q3. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q4. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q5. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q6. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

Q7. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q8. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q9. Is it possible to add a larger volume then 2 μL of enzyme to the microplate assay? In some instances 2 μL can be difficult to pipette manually.

Yes, instead of adding 2 μL of enzyme suspension an alternative is to dilute the enzyme and add a larger volume to the microplate assay.

Dilute the assay buffer 10-fold with distilled water and use this as the diluent to dilute an aliquot of the enzyme suspension also by 10-fold. Instead of 2 μL, use 20 μL of the diluted enzyme in the microplate assay.

Q10. When using this kit for quantitative analysis what level of accuracy and repeatability can be expected?

The test kit is extremely accurate – at Megazyme the quality control criteria for accuracy and repeatability is to be within 2% of the expected value using pure analytes.

However, the level of accuracy is obviously analyst and sample dependent.

Q11. Absorbance values of my sample reactions continue to increase slowly after the reaction should be complete. Is there an explanation for this?

Some samples can react with the INT in the assay and cause a non-enzymatic creep reaction.

The 3rd worksheet in the MegaCalc is used to account for any creep reaction in your results. 

Q12. Must the minimum absorbance change for a sample always be at least 0.1?

No. The 0.1 change of absorbance is only a recommendation. The lowest acceptable change in absorbance can is dictated by the analyst and equipment (i.e. pipettes and spectrophotometer) and therefore can be can be determined by the user. With accurate pipetting, absorbance changes as low as 0.02 can be used accurately.
If a change in absorbance above 0.1 is required but cannot be achieved due to low concentrations of analyte in a sample, this can be overcome by using a larger sample volume in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results. 

Q13. Can the sensitivity of the kit assay be increased?

Yes. Samples with the lower concentrations of analyte will generate a lower absorbance change. For samples with low concentrations of analyte, a larger sample volume can be used in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results.

D-异柠檬酸检测试剂盒 D-Isocitric Acid Assay Kit 货号:K-ISOC Megazyme试剂盒

D-异柠檬酸检测试剂盒

英文名:D-Isocitric Acid Assay Kit

货号:K-ISOC

规格:100 assays (manual) /

市场价: 2862

The D-Isocitric Acid test kit is for the specific and rapid measurement and analysis of D-isocitric acid in fruit juices.
Suitable for manual, auto-analyser and microplate formats.

UV-method for the determination of D-Isocitric Acid in foodstuffs

Principle:
(isocitrate dehydrogenase)
(1) D-Isocitric acid + NADP+ → 2-oxoglutarate + CO2 + NADPH + H+

(pH 9-10)
(2) D-Isocitric acid ester + H2O → D-isocitric acid + alcohol

(pH 9-10)
(3) D-Isocitric acid lactone + H2O → D-isocitric acid

Kit size: 100 assays (manual) / 1000 (microplate)
/ 1000 (auto-analyser)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 3 min
Detection limit: 0.35 mg/L
Application examples:
Fruit juices, fruit products, soft drinks and other materials (e.g. biological
cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by EN, NEN, NF,
DIN, GOST, IFU and AIJN

Advantages

  • Very competitive price (cost per test)
  • All reagents stable for > 2 years after preparation
  • No wasted isocitrate dehydrogenase solution (stable suspension supplied)
  • Very rapid reaction
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats

Q1. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q2. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q3. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q4. Does citric acid interfere with the K-ISOC assay?

Citric acid levels of at least 4 g/L (400 micrograms in a 0.1 mL sample) do not interfere with the K-ISOC test, even higher levels of citric acid can be tolerated.

Q5. Is the D-Isocitric Acid Assay Kit (K-ISOC) specific for isocitric acid in the presence of citric acid?

Yes, the D-Isocitric Acid Assay Kit (K-ISOC) is specific for isocitric acid in the presence of citric acid and at least up to Isocitric acid : citric acid ratios of 1:10.

Q6. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q7. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q8. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q9. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q10. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q11. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

Q12. Can K-ISOC be used to measure isocitric acid-lactone and if so what preparation method is used to convert it to isocitric acid?

This is a method to prepare DL-isocitric acid from DL isocitric acid-lactone but can also be used to convert DL isocitric acid-lactone to DL isocitric acid in various samples.

To prepare 0.05 M DL isocitric acid: add 435 mg DL isocitric acid-lactone into 10 mL distilled water and pH to 9 using 1 M KOH. Heat in a boiling water bath for 10 min keeping the pH above 7. Cool and adjust to pH 7.4 with 2 M HCl and dilute to 25 mL. 

Q13. When using this kit for quantitative analysis what level of accuracy and repeatability can be expected?

The test kit is extremely accurate – at Megazyme the quality control criteria for accuracy and repeatability is to be within 2% of the expected value using pure analytes.

However, the level of accuracy is obviously analyst and sample dependent.

Q14. Is it possible to add a larger volume then 2 μL of enzyme to the microplate assay? In some instances 2 μL can be difficult to pipette manually.

Yes, instead of adding 2 μL of enzyme suspension an alternative is to dilute the enzyme and add a larger volume to the microplate assay.

Dilute the assay buffer 10-fold with distilled water and use this as the diluent to dilute an aliquot of the enzyme suspension also by 10-fold. Instead of 2 μL, use 20 μL of the diluted enzyme in the microplate assay.

Q15. Can the sensitivity of the kit assay be increased?

Yes. Samples with the lower concentrations of analyte will generate a lower absorbance change. For samples with low concentrations of analyte, a larger sample volume can be used in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results.

Q16. Must the minimum absorbance change for a sample always be at least 0.1?

No. The 0.1 change of absorbance is only a recommendation. The lowest acceptable change in absorbance can is dictated by the analyst and equipment (i.e. pipettes and spectrophotometer) and therefore can be can be determined by the user. With accurate pipetting, absorbance changes as low as 0.02 can be used accurately.
If a change in absorbance above 0.1 is required but cannot be achieved due to low concentrations of analyte in a sample, this can be overcome by using a larger sample volume in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results. 

L-乳酸检测试剂盒 L-Lactic Acid (L-Lactate) Assay Kit 货号:K-LATE Megazyme试剂盒

L-乳酸检测试剂盒

英文名:L-Lactic Acid (L-Lactate) Assay Kit

货号:K-LATE

规格:50 assays (manual) / 500 assays

市场价: 2226

分析物意义:水果、蔬菜和蛋产品的质量指标  

Megazyme检测试剂盒优点:反应快、试剂稳定。适用于手工和自动分析仪进行检测 

The L-Lactic Acid (L-Lactate) Assay Kit is used for the specific measurement and analysis of L-lactic acid (L-lactate) in beverages, meat, dairy and food products.
Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
Suitable for manual, auto-analyser and microplate formats.

UV-method for the determination of L-Lactic Acid in foodstuffs,
beverages and other materials

Principle:
(L-lactate dehydrogenase)
(1) L-Lactic acid + NAD+ ↔ pyruvate + NADH + H+

(glutamate-pyruvate transaminase)
(2) Pyruvate + D-glutamate → D-alanine + 2-oxoglutarate

Kit size: 50 assays (manual) / 450 (microplate)
/ 500 (auto-analyser)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 10 min
Detection limit: 0.21 mg/L
Application examples:
Wine, beer, soft drinks, milk, dairy products (e.g. cream, milk / whey
powder, cheese, condensed milk and yogurt), foods containing milk
(e.g. dietetic foods, bakery products, baby food, chocolate, sweets
and ice-cream), egg, egg products (e.g. egg powder), baking additives,
vinegar, fruit and vegetables, processed fruit and vegetables
(e.g. tomatoes), meat products, food additives, feed, paper (and
cardboard), cosmetics, pharmaceuticals and other materials (e.g. biological
cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by DIN, GOST,
IDF, EEC, EN, ISO, OIV, IFU, AIJN and MEBAK

Advantages

  • Very competitive price (cost per test)
  • All reagents stable for > 2 years after preparation
  • Rapid reaction
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Extended cofactors stability
  • Suitable for manual, microplate and auto-analyser formats

Q1. Is the L-Lactic Acid (L-Lactate) Assay Kit (K-LATE) suitable for measurement using cell culture media samples?

Yes, assuming that the concentration of the analyte in the sample (after sample preparation) is above the limit of detection for the kit.  It may be sufficient to use the sample directly in the assay after clarification by centrifugation / filtering followed, by dilution (if required) in distilled water. 

Q2. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q3. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q4. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q5. Can perchloric acid be used to deproteinise / clarify samples prior to analysis using the L-Lactic Acid Assay Kit (K-LATE)? If so, how should such an extraction be performed?

Yes.  Perchloric acid extraction can be used in conjunction with this kit, and should be performed as follows:
WARNING: If you have not worked with perchloric acid before, you must consult your safety officer for advice.  Also, depending on the nature of the samples, it may be possible to reduce the concentration of perchloric acid, to for example 0.3 M (i.e. in the case of plasma).  It is thus very important to determine if this is possible for each type of sample used, in order to reduce the risk from working with concentrated perchloric acid.

Liquid samples:

  1. Carefully add an equal volume of ice cold 3 M perchloric acid and homogenise / fully disperse the sample (as appropriate).
  2. After 15 min incubation on ice (or in a refrigerator), centrifuge at 3000 x g for 15 min at 4˚C.
  3. Neutralise by the slow addition of 2 M KOH.
  4. Incubate on ice (or in a refrigerator) until the potassium perchlorate has settled out by gravity (approximately 10 min), and then simply remove some of the clear supernatant and use directly in the assay.

 

Solid samples:

  1. Accurately weigh approx. 5 g of homogenised sample into a beaker containing 20 mL of 1 M perchloric acid and very carefully homogenise with an Ultraturrax®  (or equivalent) for 5 min.
  2. Carefully add approx. 40 mL of distilled water and neutralise using 2 M KOH (using pH test strips for example).  Quantitatively transfer the contents to a 100 mL volumetric flask and fill to the mark with distilled water.  If a fat layer develops, make sure this is above the mark, and the aqueous layer is at the mark.
  3. Incubate in a refrigerator for approx. 20 min to allow separation of fat and precipitation of potassium perchlorate.
  4. Filter through Whatman No. 1 filter paper, discarding the first few mL of filtrate, and use directly in the assay.

Q6. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q7. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q8. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the 
K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch 
this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q9. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q10. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q11. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q12. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q1

Megazyme L-乳酸检测试剂盒操作视频(K-LATE)

Megazyme 溶解淀粉 操作视频

Megazyme 试剂盒样品前处理准备操作视频

L-苹果酸检测试剂盒 L-Malic Acid Assay Kit (Manual Format) 货号:K-LMAL-116A Megazyme试剂盒

L-苹果酸检测试剂盒

英文名:L-Malic Acid Assay Kit (Manual Format)

货号:K-LMAL-116A

规格:116 assays per kit

市场价: 3604

L-Malic Acid (Regular) Assay Kit, for the specific assay of L-malic acid (L-malate) in beverages and food products.

Manual format UV-method for the determination of L-Malic Acid in foodstuffs, beverages and other materials

Principle:
(L-malate dehydrogenase)
(1) L-Malic acid + NAD+ ↔ oxaloacetate + NADH + H+

(glutamate-oxaloacetate transaminase)
(2) Oxaloacetate + L-glutamate → L-aspartate + 2-oxoglutarate

Kit size: (K-LMALR)
58 assays (manual) / 580 (microplate) or
(K-LMALL)
116 assays (manual) / 1160 (microplate)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 3 min
Detection limit: 0.25 mg/L
Application examples:
Wine, beer, fruit juices, soft drinks, candies, fruit and vegetables,
bread, cosmetics, pharmaceuticals and other materials (e.g. biological
cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by AOAC, EEC,
EN, NF, NEN, DIN, GOST, OIV, IFU, AIJN and MEBAK

Advantages

  • PVP incorporated to prevent tannin inhibition
  • Both enzymes supplied as stable suspensions
  • Very competitive price (cost per test)
  • All reagents stable for > 2 years after preparation
  • Very rapid reaction (~ 3 min)
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Extended cofactors stability
  • Suitable for manual and microplate format

Q1. What is the difference between K-LMAL-58A / 116A, K-LMALAF, K-LMALMQ and K-LMALQR?

Megazyme produces 4 L-malic acid test kits:
K-LMAL-58A / 116A: UV method, automated format for use with auto-analysers.
K-LMALAF: UV method, manual format for use with spectrophotometers.
K-LMALMQ: Colourimetric method, manual format for use with hand held colorimeter.
K-LMALQR: UV method, liquid ready reagents automated format for use with auto-analysers.

Q2. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q3. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q4. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q5. Which L-Malic Acid Kit is recommended for a 96-well microplate format?

Auto-analysers use ~ 0.315 mL reaction volumes and pathlengths between 4-8 mm which is similar to a standard 96-well microplate where a 0.315 mL reaction volume would give a pathlength of ~ 6-7 mm.  Therefore, K-LMALAF can be used directly in a 96-well microplate format with minimal assay optimisation.
If preferred, K-LMAL-58A / 116A may also be easily converted for use in a 96-well microplate format.  Basically, the assay volumes for the cuvette format must be reduced approximately 10-fold for use in a 96-well microplate.  However, some assay optimisation may be required (e.g. increased enzyme concentration etc.) and unlike the cuvette which has a set pathlength of 1 cm, the pathlength in the microplate is dependent upon the volume of liquid in the well.
Therefore to enable the calculation of the amount of analyte in the samples from tests performed in the microplate format one of the following must be done:

  1. The easiest method is to use a microplate reader that has a pathlength conversion capability (i.e. the microplate reader can detect the pathlength of each well and convert the individual readings to a 1 cm pathlength).  This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm pathlength) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values.

L-Malic Acid Kit Recommendation For Microplate Format:
Either K-LMAL-58A / 116A or K-LMALAF is recommended for use in a 96-well microplate format and the main advantages / disadvantages are described below:
K-LMAL-58A / 116A:
The assay volumes of this kit should be reduced by 10-fold for use in a 96-well microplate format (some assay optimisation may be required, e.g. increased enzyme concentration etc.).
The calculation of results is achieved as outlined above in either of points 1, 2 or 3.
The main advantage here is that if this kit is used with a microplate reader that has a pathlength conversion capability, or if results are converted as outlined above in point 3, then this enables easy calculation of results using the K-LMAL-58A / 116A MegaCalc application (available on the Megazyme website where the product is located).
K-LMALAF:
This kit is designed for use in an auto-analyser and therefore can be used without any modification to assay volumes directly in a 96-well microplate format. 
This kit has less reagent additions than K-LMAL-58A / 116A.
K-LMALAF does not have a MegaCalc application available to enable easy results calculation which therefore must be achieved as outlined above in either of points 2 or 3.

Q6. Do samples require any specific sample preparation prior to testing with the kits?

The sample preparation is sample dependent, some samples may be tested directly in the assay or after appropriate dilution, however, some samples may require further sample preparation prior to testing.  The following are example of sample preparation methods:
(a) Liquid samples: clear, slightly coloured and approximately neutral, liquid samples can be used directly in the assay.
(b) Acidic samples: if > 0.1 mL of an acidic sample is to be used undiluted (such as wine or fruit juice), the pH of the solution should be increased to approx. 9.0 using 2 M NaOH, and the solution incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing significant quantities of carbon dioxide, such as beer, should be degassed by increasing the pH to approx. 9.0 with 2 M NaOH and gentle stirring, or by stirring with a glass rod.
(d) Coloured samples: an additional sample blank, i.e. sample with no L-MDH, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly coloured samples should be treated by the addition of 0.2 g of PVPP/10 mL of sample.  Shake the tube vigorously for 5 min and then filter through Whatman No. 1 filter paper.
(f) Solid samples: homogenise or crush solid samples in distilled water and filter if necessary.
(g) Samples containing fat: extract such samples with hot water at a temperature above the melting point of the fat, e.g. in a 100 mL volumetric flask.  Adjust to room temperature and fill the volumetric flask to the mark with distilled water.  Store on ice or in a refrigerator for 15-30 min and then filter.  Discard the first few mL of filtrate, and use the clear supernatant (which may be slightly opalescent) for assay. Alternatively, clarify with Carrez reagents.
(h) Samples containing protein: deproteinise samples containing protein by adding an equal volume of ice-cold 1 M perchloric acid with mixing.  Centrifuge at 1,500 g for 10 min and neutralise the supernatant with 1 M KOH.  Alternatively use Carrez reagents.

Q7. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the 
K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch 
this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q8. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

<h3 style="border-bottom: 0px; border-left: 0px; padding-bottom: 0px; line-height: 1.38em; font-style: normal; margin: 0px; outline-style: none; outline-color: invert; padding-left: 0px; outline-width: 0px; padding-right: 0px; font-family: proxima-nova-condensed-n4, proxima-nova-condensed, sans-serif; color: rgb(102,102,102); font-size: 13px; vertical-align: baseline; border-top: 0px; font-weight: 700 !im

Megazyme L-苹果酸(手动)检测试剂盒操作视频(K-LMAL)

Megazyme 溶解淀粉 操作视频

Megazyme 试剂盒样品前处理准备操作视频

L-苹果酸检测试剂盒 L-Malic Acid Assay Kit (Manual Format) 货号:K-LMAL-58A Megazyme试剂盒

L-苹果酸检测试剂盒

英文名:L-Malic Acid Assay Kit (Manual Format)

货号:K-LMAL-58A

规格:58 assays per kit

市场价: 2000

L-Malic Acid (Regular) Assay Kit, for the specific assay of L-malic acid (L-malate) in beverages and food products.

Manual format UV-method for the determination of L-Malic Acid in foodstuffs, beverages and other materials

Principle:
(L-malate dehydrogenase)
(1) L-Malic acid + NAD+ ↔ oxaloacetate + NADH + H+

(glutamate-oxaloacetate transaminase)
(2) Oxaloacetate + L-glutamate → L-aspartate + 2-oxoglutarate

Kit size: (K-LMALR)
58 assays (manual) / 580 (microplate) or
(K-LMALL)
116 assays (manual) / 1160 (microplate)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 3 min
Detection limit: 0.25 mg/L
Application examples:
Wine, beer, fruit juices, soft drinks, candies, fruit and vegetables,
bread, cosmetics, pharmaceuticals and other materials (e.g. biological
cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by AOAC, EEC,
EN, NF, NEN, DIN, GOST, OIV, IFU, AIJN and MEBAK

Advantages

  • PVP incorporated to prevent tannin inhibition
  • Both enzymes supplied as stable suspensions
  • Very competitive price (cost per test)
  • All reagents stable for > 2 years after preparation
  • Very rapid reaction (~ 3 min)
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Extended cofactors stability
  • Suitable for manual and microplate format

Q1. What is the difference between K-LMAL-58A / 116A, K-LMALAF, K-LMALMQ and K-LMALQR?

Megazyme produces 4 L-malic acid test kits:
K-LMAL-58A / 116A: UV method, automated format for use with auto-analysers.
K-LMALAF: UV method, manual format for use with spectrophotometers.
K-LMALMQ: Colourimetric method, manual format for use with hand held colorimeter.
K-LMALQR: UV method, liquid ready reagents automated format for use with auto-analysers.

Q2. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q3. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q4. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q5. Which L-Malic Acid Kit is recommended for a 96-well microplate format?

Auto-analysers use ~ 0.315 mL reaction volumes and pathlengths between 4-8 mm which is similar to a standard 96-well microplate where a 0.315 mL reaction volume would give a pathlength of ~ 6-7 mm.  Therefore, K-LMALAF can be used directly in a 96-well microplate format with minimal assay optimisation.
If preferred, K-LMAL-58A / 116A may also be easily converted for use in a 96-well microplate format.  Basically, the assay volumes for the cuvette format must be reduced approximately 10-fold for use in a 96-well microplate.  However, some assay optimisation may be required (e.g. increased enzyme concentration etc.) and unlike the cuvette which has a set pathlength of 1 cm, the pathlength in the microplate is dependent upon the volume of liquid in the well.
Therefore to enable the calculation of the amount of analyte in the samples from tests performed in the microplate format one of the following must be done:

  1. The easiest method is to use a microplate reader that has a pathlength conversion capability (i.e. the microplate reader can detect the pathlength of each well and convert the individual readings to a 1 cm pathlength).  This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm pathlength) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values.

L-Malic Acid Kit Recommendation For Microplate Format:
Either K-LMAL-58A / 116A or K-LMALAF is recommended for use in a 96-well microplate format and the main advantages / disadvantages are described below:
K-LMAL-58A / 116A:
The assay volumes of this kit should be reduced by 10-fold for use in a 96-well microplate format (some assay optimisation may be required, e.g. increased enzyme concentration etc.).
The calculation of results is achieved as outlined above in either of points 1, 2 or 3.
The main advantage here is that if this kit is used with a microplate reader that has a pathlength conversion capability, or if results are converted as outlined above in point 3, then this enables easy calculation of results using the K-LMAL-58A / 116A MegaCalc application (available on the Megazyme website where the product is located).
K-LMALAF:
This kit is designed for use in an auto-analyser and therefore can be used without any modification to assay volumes directly in a 96-well microplate format. 
This kit has less reagent additions than K-LMAL-58A / 116A.
K-LMALAF does not have a MegaCalc application available to enable easy results calculation which therefore must be achieved as outlined above in either of points 2 or 3.

Q6. Do samples require any specific sample preparation prior to testing with the kits?

The sample preparation is sample dependent, some samples may be tested directly in the assay or after appropriate dilution, however, some samples may require further sample preparation prior to testing.  The following are example of sample preparation methods:
(a) Liquid samples: clear, slightly coloured and approximately neutral, liquid samples can be used directly in the assay.
(b) Acidic samples: if > 0.1 mL of an acidic sample is to be used undiluted (such as wine or fruit juice), the pH of the solution should be increased to approx. 9.0 using 2 M NaOH, and the solution incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing significant quantities of carbon dioxide, such as beer, should be degassed by increasing the pH to approx. 9.0 with 2 M NaOH and gentle stirring, or by stirring with a glass rod.
(d) Coloured samples: an additional sample blank, i.e. sample with no L-MDH, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly coloured samples should be treated by the addition of 0.2 g of PVPP/10 mL of sample.  Shake the tube vigorously for 5 min and then filter through Whatman No. 1 filter paper.
(f) Solid samples: homogenise or crush solid samples in distilled water and filter if necessary.
(g) Samples containing fat: extract such samples with hot water at a temperature above the melting point of the fat, e.g. in a 100 mL volumetric flask.  Adjust to room temperature and fill the volumetric flask to the mark with distilled water.  Store on ice or in a refrigerator for 15-30 min and then filter.  Discard the first few mL of filtrate, and use the clear supernatant (which may be slightly opalescent) for assay. Alternatively, clarify with Carrez reagents.
(h) Samples containing protein: deproteinise samples containing protein by adding an equal volume of ice-cold 1 M perchloric acid with mixing.  Centrifuge at 1,500 g for 10 min and neutralise the supernatant with 1 M KOH.  Alternatively use Carrez reagents.

Q7. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the 
K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch 
this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q8. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

<h3 style="border-bottom: 0px; border-left: 0px; padding-bottom: 0px; line-height: 1.38em; font-style: normal; margin: 0px; outline-style: none; outline-color: invert; padding-left: 0px; outline-width: 0px; padding-right: 0px; font-family: proxima-nova-condensed-n4, proxima-nova-condensed, sans-serif; color: rgb(102,102,102); font-size: 13px; vertical-align: baseline; border-top: 0px; font-weight: 700 !im

Megazyme L-苹果酸(手动)检测试剂盒操作视频(K-LMAL)

Megazyme 溶解淀粉 操作视频

Megazyme 试剂盒样品前处理准备操作视频

L-苹果酸[AF]检测试剂盒 L-Malic Acid Assay Kit (Analyser Format) 货号:K-LMALAF Megazyme试剂盒

L-苹果酸[AF]检测试剂盒

英文名:L-Malic Acid Assay Kit (Analyser Format)

货号:K-LMALAF

规格:245.5 mL of prepared reagent (e.g. 1 assay

市场价: 3710

The L-Malic Acid (Analyser Format) test kit is an analyser format for the specific measurement and analysis of L-malic acid (L-malate) in beverages and food products. On calibration, the prepared reagent is linear to > 80 micrograms of L-malic acid per mL of assay solution.

Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.

Analyser format UV-method for the determination of L-Malic Acid
in foodstuffs, beverages and other materials

Principle:
(L-malate dehydrogenase)
(1) L-Malic acid + NAD+ ↔ oxaloacetate + NADH + H+

(glutamate-oxaloacetate transaminase)
(2) Oxaloacetate + L-glutamate → L-aspartate + 2-oxoglutarate

Kit size: 245.5 mL of prepared reagent (R1 + R2)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 3 min
Detection limit: 20 mg/L (recommended assay)
Application examples:
Wine, beer, fruit juices, soft drinks, candies, fruit and vegetables,
bread, cosmetics, pharmaceuticals and other materials (e.g. biological
cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by AOAC, EEC,
EN, NF, NEN, DIN, GOST, OIV, IFU, AIJN and MEBAK

Advantages

  • PVP incorporated to prevent tannin inhibition
  • Very stable reagent when prepared for auto-analyser applications
  • Linear calibration (R2 ~ 0.9994) up to 80 μg/mL of L-malic acid in final reaction solution
  • Validated by the University of Wine, Suze la Rousse, France
  • Very competitive price (cost per mL of reagent)
  • Both enzymes supplied as stable suspensions
  • Very rapid reaction (~ 3 min)

Q1. What is the difference between K-LMAL-58A / 116A, K-LMALAF, K-LMALMQ and K-LMALQR?

Megazyme produces 4 L-malic acid test kits:
K-LMAL-58A / 116A: UV method, automated format for use with auto-analysers.
K-LMALAF: UV method, manual format for use with spectrophotometers.
K-LMALMQ: Colourimetric method, manual format for use with hand held colorimeter.
K-LMALQR: UV method, liquid ready reagents automated format for use with auto-analysers.

Q2. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q3. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q4. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q5. Do samples require any specific sample preparation prior to testing with the kits?

The sample preparation is sample dependent, some samples may be tested directly in the assay or after appropriate dilution, however, some samples may require further sample preparation prior to testing.  The following are examples of sample preparation methods:
(a) Liquid samples: clear, slightly coloured and approximately neutral, liquid samples can be used directly in the assay.
(b) Acidic samples: if > 0.1 mL of an acidic sample is to be used undiluted (such as wine or fruit juice), the pH of the solution should be increased to approx. 9.0 using 2 M NaOH, and the solution incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing significant quantities of carbon dioxide, such as beer, should be degassed by increasing the pH to approx. 9.0 with 2 M NaOH and gentle stirring, or by stirring with a glass rod.
(d) Coloured samples: an additional sample blank, i.e. sample with no L-MDH, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly coloured samples should be treated by the addition of 0.2 g of PVPP/10 mL of sample.  Shake the tube vigorously for 5 min and then filter through Whatman No. 1 filter paper.
(f) Solid samples: homogenise or crush solid samples in distilled water and filter if necessary.
(g) Samples containing fat: extract such samples with hot water at a temperature above the melting point of the fat, e.g. in a 100 mL volumetric flask.  Adjust to room temperature and fill the volumetric flask to the mark with distilled water.  Store on ice or in a refrigerator for 15-30 min and then filter.  Discard the first few mL of filtrate, and use the clear supernatant (which may be slightly opalescent) for assay. Alternatively, clarify with Carrez reagents.
(h) Samples containing protein: deproteinise samples containing protein by adding an equal volume of ice-cold 1 M perchloric acid with mixing.  Centrifuge at 1,500 g for 10 min and neutralise the supernatant with 1 M KOH. Alternatively use Carrez reagents.

Q6. Which L-Malic Acid Kit is recommended for a 96-well microplate format?

Auto-analysers use ~ 0.315 mL reaction volumes and pathlengths between 4-8 mm which is similar to a standard 96-well microplate where a 0.315 mL reaction volume would give a pathlength of ~ 6-7 mm.  Therefore, K-LMALAF can be used directly in a 96-well microplate format with minimal assay optimisation.
If preferred, K-LMAL-58A / 116A may also be easily converted for use in a 96-well microplate format.  Basically, the assay volumes for the cuvette format must be reduced approximately 10-fold for use in a 96-well microplate.  However, some assay optimisation may be required (e.g. increased enzyme concentration etc.) and unlike the cuvette which has a set pathlength of 1 cm, the pathlength in the microplate is dependent upon the volume of liquid in the well.
Therefore to enable the calculation of the amount of analyte in the samples from tests performed in the microplate format one of the following must be done:

  1. The easiest method is to use a microplate reader that has a pathlength conversion capability (i.e. the microplate reader can detect the pathlength of each well and convert the individual readings to a 1 cm pathlength).  This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm pathlength) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values.

L-Malic Acid Kit Recommendation For Microplate Format:
Either K-LMAL-58A / 116A or K-LMALAF is recommended for use in a 96-well microplate format and the main advantages / disadvantages are described below:
K-LMAL-58A / 116A:
The assay volumes of this kit should be reduced by 10-fold for use in a 96-well microplate format (some assay optimisation may be required, e.g. increased enzyme concentration etc.).
The calculation of results is achieved as outlined above in either of points 1, 2 or 3.
The main advantage here is that if this kit is used with a microplate reader that has a pathlength conversion capability or if results are converted as outlined above in point 3 then this enables easy calculation of results using the K-LMAL-58A / 116A MegaCalc application (available on the Megazyme website where the product is located).
K-LMALAF:
This kit is designed for use in an auto-analyser and therefore can be used without any modification to assay volumes directly in a 96-well microplate format.  This kit has less reagent additions than K-LMAL-58A / 116A. 
K-LMALAF does not have a MegaCalc application available to enable easy results calculation which therefore must be achieved as outlined above in either of points 2 or 3.

Q7. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the 
K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch 
this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q8. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q9. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

L-苹果酸[MegaQuant法]检测试剂盒 L-Malic Acid Assay Kit (MegaQuant™ Format) 货号:K-LMALMQ Megazyme试剂盒

L-苹果酸[MegaQuant法]检测试剂盒

英文名:L-Malic Acid Assay Kit (MegaQuant™ Format)

货号:K-LMALMQ

规格:60 assays per kit.

市场价: 3200

The L-Malic Acid test kit is suitable for the measurement and analysis of L-malic acid in grapes, grape juice and wine using the MegaQuant™ colorimeter (measurement at 505 nm). Suitable for white and red wines at all stages of the winemaking process.

Simple colourimetric method for the determination of L-Malic
Acid in foodstuffs, beverages and other materials

Principle:
(L-malate dehydrogenase)
(1) L-Malic acid + NAD+ ↔ oxaloacetate + NADH + H+

(glutamate-oxaloacetate transaminase)
(2) Oxaloacetate + L-glutamate → L-aspartate + 2-oxoglutarate

(diaphorase)
(3) INT + NADH + H+ → NAD+ + INT-formazan

Kit size: 60 assays
Method: Spectrophotometric at 505 nm
Reaction time: ~ 6 min
Detection limit: 0.25 mg/L
Application examples:
Wine, beer, fruit juices, soft drinks, candies, fruit and vegetables,
bread, cosmetics, pharmaceuticals and other materials (e.g. biological
cultures, samples, etc.)
Method recognition: Novel method

Advantages

  • Novel product, patented technology
  • Highly stable reagents (at least three seasons use)
  • Very competitive price (cost per test)
  • Spectrophotometer / laboratory / expertise not required
  • Very simple procedure
  • Rapid reaction time (~6 min)
  • Standard included

 

Q1. What is the difference between K-LMAL-58A/116A, K-LMALAF, K-LMALMQ and K-LMALQR?

Megazyme produces 4 L-malic acid test kits:
K-LMAL-58A / 116A: UV method, automated format for use with auto-analysers.
K-LMALAF: UV method, manual format for use with spectrophotometers.
K-LMALMQ: Colourimetric method, manual format for use with hand held colorimeter.
K-LMALQR: UV method, liquid ready reagents automated format for use with auto-analysers.

Q2. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q3. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q4. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q5. Which L-Malic Acid Kit is recommended for a 96-well microplate format?

Auto-analysers use ~ 0.315 mL reaction volumes and pathlengths between 4-8 mm which is similar to a standard 96-well microplate where a 0.315 mL reaction volume would give a pathlength of ~ 6-7 mm.  Therefore, K-LMALAF can be used directly in a 96-well microplate format with minimal assay optimisation.
If preferred, K-LMAL-58A / 116A may also be easily converted for use in a 96-well microplate format.  Basically, the assay volumes for the cuvette format must be reduced approximately 10-fold for use in a 96-well microplate.  However, some assay optimisation may be required (e.g. increased enzyme concentration etc.) and unlike the cuvette which has a set pathlength of 1 cm, the pathlength in the microplate is dependent upon the volume of liquid in the well.
Therefore to enable the calculation of the amount of analyte in the samples from tests performed in the microplate format one of the following must be done:

  1. The easiest method is to use a microplate reader that has a pathlength conversion capability (i.e. the microplate reader can detect the pathlength of each well and convert the individual readings to a 1 cm pathlength).  This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm pathlength) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values.

L-Malic Acid Kit Recommendation For Microplate Format:
Either K-LMAL-58A / 116A or K-LMALAF is recommended for use in a 96-well microplate format and the main advantages / disadvantages are described below:
K-LMAL-58A / 116A:
The assay volumes of this kit should be reduced by 10-fold for use in a 96-well microplate format (some assay optimisation may be required, e.g. increased enzyme concentration etc.).
The calculation of results is achieved as outlined above in either of points 1, 2 or 3.
The main advantage here is that if this kit is used with a microplate reader that has a pathlength conversion capability, or if results are converted as outlined above in point 3, then this enables easy calculation of results using the K-LMAL-58A / 116A MegaCalc application (available on the Megazyme website where the product is located).
K-LMALAF:
This kit is designed for use in an auto-analyser and therefore can be used without any modification to assay volumes directly in a 96-well microplate format. 
This kit has less reagent additions than K-LMAL-58A / 116A.
K-LMALAF does not have a MegaCalc application available to enable easy results calculation which therefore must be achieved as outlined above in either of points 2 or 3.

Q6. Do samples require any specific sample preparation prior to testing with the kits?

The sample preparation is sample dependent, some samples may be tested directly in the assay or after appropriate dilution, however, some samples may require further sample preparation prior to testing.  The following are example of sample preparation methods:
(a) Liquid samples: clear, slightly coloured and approximately neutral, liquid samples can be used directly in the assay.
(b) Acidic samples: if > 0.1 mL of an acidic sample is to be used undiluted (such as wine or fruit juice), the pH of the solution should be increased to approx. 9.0 using 2 M NaOH, and the solution incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing significant quantities of carbon dioxide, such as beer, should be degassed by increasing the pH to approx. 9.0 with 2 M NaOH and gentle stirring, or by stirring with a glass rod.
(d) Coloured samples: an additional sample blank, i.e. sample with no L-MDH, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly coloured samples should be treated by the addition of 0.2 g of PVPP/10 mL of sample.  Shake the tube vigorously for 5 min and then filter through Whatman No. 1 filter paper.
(f) Solid samples: homogenise or crush solid samples in distilled water and filter if necessary.
(g) Samples containing fat: extract such samples with hot water at a temperature above the melting point of the fat, e.g. in a 100 mL volumetric flask.  Adjust to room temperature and fill the volumetric flask to the mark with distilled water.  Store on ice or in a refrigerator for 15-30 min and then filter.  Discard the first few mL of filtrate, and use the clear supernatant (which may be slightly opalescent) for assay. Alternatively, clarify with Carrez reagents.
(h) Samples containing protein: deproteinise samples containing protein by adding an equal volume of ice-cold 1 M perchloric acid with mixing.  Centrifuge at 1,500 g for 10 min and neutralise the supernatant with 1 M KOH.  Alternatively use Carrez reagents.

Q7. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q8. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q9. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the 
K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch 
this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q10. I have some doubts about the appearance/quality of a kit component what should be done?

L-苹果酸[液体即用型]检测试剂盒 L-Malic Acid Assay Kit (Liquid Ready Reagents) 货号:K-LMALQR Megazyme试剂盒

L-苹果酸[液体即用型]检测试剂盒

英文名:L-Malic Acid Assay Kit (Liquid Ready Reagents)

货号:K-LMALQR

规格:1100 assays (microplate) / 1100 (auto-analyser)

市场价: 2862

The L-Malic Acid (Liquid Ready Reagents) test kit is a rapid, simple, reliable and accurate method for the specific measurement and analysis of L-malic acid in wine, beverages, foodstuffs and other materials. Supplied as a “ready to use” liquid stable formulation that is suitable for auto-analyser and microplate formats.
Suitable for auto-analyser and microplate formats.

UV-method suitable for microplate and auto-analyser formats
for the determination of L-Malic Acid in foodstuffs, beverages
and other materials

Principle:
(L-malate dehydrogenase)
(1) L-Malic acid + NAD+ ↔ oxaloacetate + NADH + H+

(glutamate-oxaloacetate transaminase)
(2) Oxaloacetate + L-glutamate → L-aspartate + 2-oxoglutarate

Kit size: 1100 assays (microplate)
/ 1100 (auto-analyser)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 3 min
Detection limit: 166 mg/L (recommended format)
Application examples:
Wine, beer, fruit juices, soft drinks, candies, fruit and vegetables,
bread, cosmetics, pharmaceuticals and other materials (e.g. biological
cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by AOAC, EEC,
EN, NF, NEN, DIN, GOST, OIV, IFU, AIJN and MEBAK

Advantages

  • PVP incorporated to prevent tannin inhibition
  • “Ready to use” liquid stable formulation
  • Very competitive price (cost per test)
  • All reagents stable for > 18 Months
  • Very rapid reaction (~ 3 min)
  • Standard included
  • Suitable for microplate and auto-analyser formats

Q1. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q2. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q3. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q4. What is the difference between K-LMAL-58A / 116A, K-LMALAF, K-LMALMQ and K-LMALQR?

Megazyme produces 4 L-malic acid test kits:
K-LMAL-58A / 116A: UV method, automated format for use with auto-analysers.
K-LMALAF: UV method, manual format for use with spectrophotometers.
K-LMALMQ: Colourimetric method, manual format for use with hand held colorimeter.
K-LMALQR: UV method, liquid ready reagents automated format for use with auto-analysers.

Q5. Which L-Malic Acid Kit is recommended for a 96-well microplate format?

Auto-analysers use ~ 0.315 mL reaction volumes and pathlengths between 4-8 mm which is similar to a standard 96-well microplate where a 0.315 mL reaction volume would give a pathlength of ~ 6-7 mm.  Therefore, K-LMALAF can be used directly in a 96-well microplate format with minimal assay optimisation.
If preferred, K-LMAL-58A / 116A may also be easily converted for use in a 96-well microplate format.  Basically, the assay volumes for the cuvette format must be reduced approximately 10-fold for use in a 96-well microplate. However, some assay optimisation may be required (e.g. increased enzyme concentration etc.) and unlike the cuvette which has a set pathlength of 1 cm, the pathlength in the microplate is dependent upon the volume of liquid in the well.
Therefore to enable the calculation of the amount of analyte in the samples from tests performed in the microplate format one of the following must be done:

  1. The easiest method is to use a microplate reader that has a pathlength conversion capability (i.e. the microplate reader can detect the pathlength of each well and convert the individual readings to a 1 cm pathlength).  This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm pathlength) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values.

L-Malic Acid Kit Recommendation For Microplate Format:
Either K-LMAL-58A / 116A or K-LMALAF is recommended for use in a 96-well microplate format and the main advantages / disadvantages are described below:
K-LMAL-58A / 116A:
The assay volumes of this kit should be reduced by 10-fold for use in a 96-well microplate format (some assay optimisation may be required, e.g. increased enzyme concentration etc.).
The calculation of results is achieved as outlined above in either of points 1, 2 or 3.
The main advantage here is that if this kit is used with a microplate reader that has a pathlength conversion capability, or if results are converted as outlined above in point 3, then this enables easy calculation of results using the K-LMAL-58A / 116A MegaCalc application (available on the Megazyme website where the product is located).
K-LMALAF:
This kit is designed for use in an auto-analyser and therefore can be used without any modification to assay volumes directly in a 96-well microplate format. 
This kit has less reagent additions than K-LMAL-58A / 116A.
K-LMALAF does not have a MegaCalc application available to enable easy results calculation which therefore must be achieved as outlined above in either of points 2 or 3.

Q6. Do samples require any specific sample preparation prior to testing with the kits?

The sample preparation is sample dependent, some samples may be tested directly in the assay or after appropriate dilution, however, some samples may require further sample preparation prior to testing.  The following are example of sample preparation methods:
(a) Liquid samples: clear, slightly coloured and approximately neutral, liquid samples can be used directly in the assay.
(b) Acidic samples: if > 0.1 mL of an acidic sample is to be used undiluted (such as wine or fruit juice), the pH of the solution should be increased to approx. 9.0 using 2 M NaOH, and the solution incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing significant quantities of carbon dioxide, such as beer, should be degassed by increasing the pH to approx. 9.0 with 2 M NaOH and gentle stirring, or by stirring with a glass rod.
(d) Coloured samples: an additional sample blank, i.e. sample with no L-MDH, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly coloured samples should be treated by the addition of 0.2 g of PVPP/10 mL of sample.  Shake the tube vigorously for 5 min and then filter through Whatman No. 1 filter paper.
(f) Solid samples: homogenise or crush solid samples in distilled water and filter if necessary.
(g) Samples containing fat: extract such samples with hot water at a temperature above the melting point of the fat, e.g. in a 100 mL volumetric flask.  Adjust to room temperature and fill the volumetric flask to the mark with distilled water.  Store on ice or in a refrigerator for 15-30 min and then filter.  Discard the first few mL of filtrate, and use the clear supernatant (which may be slightly opalescent) for assay. Alternatively, clarify with Carrez reagents.
(h) Samples containing protein: deproteinise samples containing protein by adding an equal volume of ice-cold 1 M perchloric acid with mixing.  Centrifuge at 1,500 g for 10 min and neutralise the supernatant with 1 M KOH.  Alternatively use Carrez reagents.

Q7. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q8. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the 
K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch 
this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q9. Can the sensitivity of the kit assay be increased?

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  • L-苹果酸[液体即用型]检测试剂盒 L-Malic Acid Assay Kit (Liquid Ready Reagents) 货号:K-LMALQR  Megazyme试剂盒 L-苹果酸[液体即用型]检测试剂盒 L-Malic Acid Assay Kit (Liquid Ready Reagents) 货号:K-LMALQR  Megazyme试剂盒 L-苹果酸[液体即用型]检测试剂盒 L-Malic Acid Assay Kit (Liquid Ready Reagents) 货号:K-LMALQR  Megazyme试剂盒 L-苹果酸[液体即用型]检测试剂盒 L-Malic Acid Assay Kit (Liquid Ready Reagents) 货号:K-LMALQR  Megazyme试剂盒 L-苹果酸[液体即用型]检测试剂盒 L-Malic Acid Assay Kit (Liquid Ready Reagents) 货号:K-LMALQR  Megazyme试剂盒
  • L-苹果酸[液体即用型]检测试剂盒 L-Malic Acid Assay Kit (Liquid Ready Reagents) 货号:K-LMALQR  Megazyme试剂盒

植酸/总磷量检测试剂盒 Phytic Acid/Total Phosphorus Assay Kit 货号:K-PHYT Megazyme试剂盒

植酸/总磷量检测试剂盒

英文名:Phytic Acid/Total Phosphorus Assay Kit

货号:K-PHYT

规格:50 assays per kit

市场价: 2968

The Phytic Acid (Total Phosphorus) test kit is a simple method for the measurement and analysis of phytic acid/total phosphorus in food and feed samples. This method does not require purification of phytic acid via anion-exchange chromatography making it amenable to high numbers of samples.

Colourimetric method for the determination of Phytic Acid
in cereal products, seed materials, animal feeds and
other materials

Principle:
(phytase)
(1) Phytic acid + H2O → myo-Inositol (phosphate)n + P i

(alkaline phosphatase)
(2) myo-Inositol (phosphate)n + H2O myo-inositol + Pi

(3) Pi + ammonium molybdate → 12-molybdophosphoric acid

(diaphorase)
(4) 12-molybdophosphoric acid + H2SO4 / ascorbic acid →
molybdenum blue

Kit size: 50 assays
Method: Spectrophotometric at 655 nm
Reaction time: 25 min enzymic; 1 h for phosphate determination
Detection limit: ~ 11.3 mg phosphorus (~ 40 mg phytic acid)
/100 g material
Application examples:
Seed materials, feeds and foodstuffs
Method recognition: Novel method

Advantages

  • Very cost effective
  • All reagents stable for > 2 years after preparation
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included

Q1. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q2. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q3. Please can you explain how the 55.6-fold dilution factor is derived.

The 55.6-fold dilution factor is the overall dilution of the sample (after extraction) throughout the Standard Assay Procedure of K-PHYT (Part A step 2, and Part B).

Q4. Can liquid samples be tested for phytic acid using K-PHYT?

Yes. Liquid samples can be tested for phytic acid using K-PHYT.

At Step 1 of the procedure (Sample Extraction), dilute a given volume of the liquid sample with an appropriate volume of hydrochloric acid (0.66 M), e.g. 1 mL liquid sample plus 19 mL hydrochloric acid (0.66 M) and proceed with the extraction incubation as for solid samples. The level of sample dilution can be adjust accordingly depending on the phytic acid content of the sample. This will need to be determined experimentally.

In the calculation for liquid samples, the sample weight (g) is replaced with sample volume (mL), e.g. if 1 mL of sample was added to 19 mL of hydrochloric acid (0.66 M) in Step 1 of the procedure (Sample Extraction) then in the calculation the sample volume is 1 and extraction volume is 20. The results of phosphorus and phytic acid content for liquid samples are given as g/100 mL instead of g/100 g.

The K-PHYT MegaCalc spreadsheet contains a separate worksheet for the calculation of results using liquid samples.

Q5. How accurate is K-PHYT for measuring unprocessed samples?

Unprocessed samples originating from grains usually contain ~ 97% of the total phosphorus content as phytic acid and therefore can be accurately measured using K-PHYT.
When analysing more pure forms of  phytic acid (inositol hexakisphosphate) (e.g. Sigma product P5681) the Phytic Acid Kit (K-PHYT) is very accurate and is able to generate results within ~ 2% of the value stated for the product after taking into account the purity and moisture content of the dry sample.

Q6. What does K-PHYT measure?

Firstly the definition of measurement should be clearly defined prior to the analyses, e.g. are the analyses for total phosphorus (this includes enzymatically released phosphorus from sources other than phytic acid), or phytic acid?
The K-PHYT Kit measures the total phosphorus released by phytase and alkaline phosphatase.  The measurement is given as grams of phosphorus / 100 g original material.
When measuring for phytic acid the assumption is that all of the phosphorus that was measured is released from phytic acid (IP6) and therefore the amount of phosphorus released is then converted to an amount of phytic acid where phosphorus comprises 28.2% of phytic acid.

Q7. How accurate is K-PHYT for measuring processed samples and does the protocol require any modifications?

When processed samples, which may contain various monophosphates not associated with phytic acid, are analysed the phytic acid content may be overestimated since the alkaline phosphatase can release phosphate from various monophosphates in addition to inositol-monophosphate and the calculation of phytic acid assumes that all of the phosphorus is released from phytic acid.  For such samples this may be overcome by performing the “Free Phosphorus” sample test exactly as described in the K-PHYT data booklet except that the alkaline phosphatase is included in the second part of the dephosphorylation reaction step (in place of the water addition).  This sample is not treated with phytase but is treated with alkaline phosphatase and will give the amount of enzymatically released phosphorous which is not associated with phytic acid.  Subtracting this value of alkaline phosphatase released phosphorus from the Total Phosphorus value for the same sample and then calculating the phytic acid would be more accurate for phytic acid determination.

Q8. What type of certificates do you give with the available samples? What are the reference values and which methods have been used to certify them?

The certificate that Megazyme currently supplies for K-PHYT is the certificate of analysis for the kit (available where the product is located on the Megazyme website).
Measurements using K-PHYT rely on accurate determination of phosphorus.  The performance of this kit requires that a phosphorus standard curve is performed for each batch of tests and the phosphorus standard that is supplied with the kit is standardised against a certified phosphorus standard obtained from SIGMA (cat no. 79409).  If required this certified standard may be used in place of the phosphorus standard that is supplied with K-PHYT.
The oat sample supplied with the kit is to be used as a control and should generate phosphorus / phytic acid values within 10% of the values supplied on the sample label.

Q9. Can solid samples be tested for phytic acid using K-PHYT?

Yes.  Solid samples such as grain can be tested for phytic acid using K-PHYT.  For solid samples, perform step 1 of the Sample Extraction using the following modification:
1. Accurately weigh approximately 1 g of sample material into a 75 mL glass beaker. Add 20 mL of hydrochloric acid (0.66 M) and homogenise the sample using a standard laboratory homogeniser or mortar and pestle, cover the beaker with foil and stir vigorously for a minimum of 3 hours at room temperature (preferably overnight for convenience).  Follow the full Standard Assay Procedure as described in the K-PHYT data booklet from step 2 of the Sample Extraction procedure.
Alternatively mill cereal, plant or food product to pass a 0.5 mm screen, then follow the full Standard Assay Procedure as described in the K-PHYT data booklet from step 1 of the Sample Extraction procedure.

Q10. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q11. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

Q12. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q13. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q14. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q15. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q16. When using this kit for quantitative analysis what level of accuracy and repeatability can be expected?

The test kit is extremely accurate – at Megazyme the quality control criteria for accuracy and repeatability is to be within 2% of the expected value using pure analytes.

However, the level of accuracy is obviously analyst and sample dependent.

Q17. Must the minimum absorbance change for a sample always be at least 0.1?

No. The 0.1 change of absorbance is only a recommendation. The lowest acceptable change in absorbance can is dictated by the analyst and equipment (i.e. pipettes and spectrophotometer) and therefore can be can be determined by the user. With accurate pipetting, absorbance changes as low as 0.02 can be used accurately.
If a change in absorbance above 0.1 is required but cannot be achieved due to low concentrations of analyte in a sample, this can be overcome by using a larger sample volume in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results. 

Q18. Can the sensitivity of the kit assay be increased?

百度云网盘下载:http://pan.baidu.com/s/1c1U019m

丙酮酸检测试剂盒 Pyruvic Acid Assay Kit 货号:K-PYRUV Megazyme试剂盒

丙酮酸检测试剂盒

英文名:Pyruvic Acid Assay Kit

货号:K-PYRUV

规格:100 assays (manual) / 1000 assays (microplate)

市场价: 2650

For the specific and rapid measurement and analysis of pyruvic acid in beer, wine, fruit juice, food products and bodily fluids.
Suitable for manual, auto-analyser and microplate formats.

UV-method for the determination of Pyruvic Acid in beer, cheese,
fermentation products and other materials

Principle:
(D-lactate dehydrogenase)
(1) Pyruvate + NADH + H+ → D-lactic acid + NAD+

Kit size: 100 assays (manual) / 1000 (microplate)
/ 1000 (auto-analyser)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 3 min
Detection limit: 0.39 mg/L
Application examples:
Wine, beer, fruit juices, soft drinks, cheese, dietary supplements,
pharmaceuticals and other materials (e.g. biological cultures,
samples, etc.)
Method recognition: New method

Advantages

  • Very cost effective
  • All reagents stable for > 2 years after preparation
  • Very rapid reaction (~ 3 min)
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats

 

Q1. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.

Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.

Samples above 0.1 mL are more likely to affect the pH of the assay solution and   therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q2. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q3. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q4. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q5. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q6. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q7. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q8. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

Q9. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q10. When using this kit for quantitative analysis what level of accuracy and repeatability can be expected?

The test kit is extremely accurate – at Megazyme the quality control criteria for accuracy and repeatability is to be within 2% of the expected value using pure analytes.

However, the level of accuracy is obviously analyst and sample dependent.

Q11. Is it possible to add a larger volume then 2 μL of enzyme to the microplate assay? In some instances 2 μL can be difficult to pipette manually.

Yes, instead of adding 2 μL of enzyme suspension an alternative is to dilute the enzyme and add a larger volume to the microplate assay.

Dilute the assay buffer 10-fold with distilled water and use this as the diluent to dilute an aliquot of the enzyme suspension also by 10-fold. Instead of 2 μL, use 20 μL of the diluted enzyme in the microplate assay.

Q12. Must the minimum absorbance change for a sample always be at least 0.1?

No. The 0.1 change of absorbance is only a recommendation. The lowest acceptable change in absorbance can is dictated by the analyst and equipment (i.e. pipettes and spectrophotometer) and therefore can be can be determined by the user. With accurate pipetting, absorbance changes as low as 0.02 can be used accurately.
If a change in absorbance above 0.1 is required but cannot be achieved due to low concentrations of analyte in a sample, this can be overcome by using a larger sample volume in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results. 

Q13. Can the sensitivity of the kit assay be increased?

Yes. Samples with the lower concentrations of analyte will generate a lower absorbance change. For samples with low concentrations of analyte, a larger sample volume can be used in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results.

琥珀酸/丁二酸检测试剂盒 Succinic Acid Assay Kit 货号:K-SUCC Megazyme试剂盒

琥珀酸/丁二酸检测试剂盒

英文名:Succinic Acid Assay Kit

货号:K-SUCC

规格:20 assays (manual) / 200 assays

市场价: 3498

The Succinic Acid test kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products.
Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
Suitable for manual, auto-analyser and microplate formats.

UV-method for the determination of Succinic Acid in foodstuffs,
feed, wine and other materials

Principle:
(succinyl-CoA synthetase)
(1) Succinic acid + ATP + CoA → ADP + succinyl-CoA + Pi

(pyruvate kinase)
(2) ADP + PEP → ATP + pyruvate

(L-lactate dehydrogenase)
(3) Pyruvate + NADH + H+ → NAD+ + L-lactic acid

Kit size: 20 assays (manual) / 200 (microplate)
/ 270 (auto-analyser)
Method: Spectrophotometric at 340 nm
Reaction time: ~ 6 min
Detection limit: 0.26 mg/L
Application examples:
Wine, fruit and vegetables, soy sauce, cheese, egg, egg products
and other materials (e.g. biological cultures, samples, etc.)
Method recognition:
Methods based on this principle have been accepted by EEC

Advantages

  • Very competitive price (cost per test)
  • All reagents stable for > 2 years as supplied
  • Very rapid reaction (even at room temperature)
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Extended cofactors stability
  • Suitable for manual, microplate and auto-analyser formats

Q1. Is the Succinic Acid Assay Kit (K-SUCC) suitable for measurement using cell culture media samples?

Yes, assuming that the concentration of the analyte in the sample (after sample preparation) is above the limit of detection for the kit.  It may be sufficient to use the sample directly in the assay after clarification by centrifugation / filtering followed by dilution (if required) in distilled water. 

Q2. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

 

Q3. Sometimes a negative absorbance change is obtained for the blank samples, is this normal? Should the real value (negative absorbance change) or “0” be used in the calculation of results?

Sometimes the addition of the last assay component can cause a small negative absorbance change in the blank samples due to a dilution effect and in such cases it is recommended that the real absorbance values be used in the calculation of results.

Q4. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q5. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q6. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q7. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the 
K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch 
this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q8. I have some doubts about the appearance/quality of a kit component what should be done?

If there are any concerns with any kit components, the first thing to do is to test the standard sample (control sample) that is supplied with the kit and ensure that the expected value (within the accepted variation) is obtained before testing any precious samples. This must be done using the procedure provided in the kit booklet without any modifications to the procedure. If there are still doubts about the results using the standard sample in the kit then send example results in the MegaCalc spread sheet to your product supplier (Megazyme or your local Megazyme distributor).

Q9. How much sample should be used for the clarification/extraction of my sample?

The volume/weight of sample and total volume of the extract can be modified to suit the sample. This will ultimately be dictated by the amount of analyte of interest in the sample and may require empirical determination. For low levels of analyte the sample:extract volume ratio can be increased (i.e. increase the sample and/or decrease the total extraction volume).

Alternatively, for samples with low concentrations of analyte, a larger sample volume can be added to the kit assay. When altering the sample volume adjust the distilled water volume added to the assay accordingly so that the total assay volume is not altered.

Q10. Can the test kit be used to measure biological fluids and what sample preparation method should be used?

The kit assay may work for biological fluids assuming that inositol is present above the limit of detection for the kit after any sample preparation (if required). Centrifugation of the samples and use of the supernatant directly in the kit assay (with appropriate dilution in distilled water) may be sufficient. However, if required a more stringent sample preparation method may be required and examples are provided at the following link:http://www.megazyme.com/docs/analytical-applications-downloads/biological_samples_111109.pdf?sfvrsn=2

The test kit has not been tested using biological fluids as samples because it is not marketed or registered as a medical device. This will therefore require your own validation.

Q11. Can the manual assay format be scaled down to a 96-well microplate format?

The majority of the Megazyme test kits are developed to work in cuvettes using the manual assay format, however the assay can be converted for use in a 96-well microplate format. To do this the assay volumes for the manual cuvette format are reduced by 10-fold. The calculation of results for the manual assay format uses a 1 cm path-length, however the path-length in the microplate is not 1 cm and therefore the MegaCalc spreadsheet or the calculation provided in the kit booklet for the manual format cannot be used for the micropalate format unless the microplate reader being used can.

There a 3 main methods for calculation of results using the microplate format:

  1. The easiest method is to use a microplate reader that has a path-length conversion capability (i.e. the microplater reader can detect the path-length of each well and convert the individual readings to a 1 cm path-length). This will allow values to be calculated using the MegaCalc calculation software which can be found where the product is located on the Megazyme website.
  2. Perform a standard curve of the analyte on each microplate that contains test samples and calculate the result of the test samples from the calibration curve (concentration of analyte versus absorbance).
  3. Perform a standard curve of the analyte in both the cuvette format (i.e. with a 1 cm path-length) and the 96-well microplate format and use these results to obtain a mean conversion factor between the cuvette values and the microplate values. Subsequent assays in the microplate format can then be converted from the calculated conversion factor.

Q12. Can the sensitivity of the kit assay be increased?

For samples with low concentrations of analyte the sample volume used in the kit assay can be increased to increase sensitivity. When doing this the water volume is adjusted to retain the same final assay volume. This is critical for the manual assay format because the assay volume and sample volume are used in the calculation of results.

Q13. Must the minimum absorbance change for a sample always be at least 0.1?

No. The 0.1 change of absorbance is only a recommendation. The lowest acceptable change in absorbance can is dictated by the analyst and equipment (i.e. pipettes and spectrophotometer) and therefore can be can be determined by the user. With accurate pipetting, absorbance changes as low as 0.02 can be used accurately.
If a change in absorbance above 0.1 is required but cannot be achieved due to low concentrations of analyte in a sample, this can be overcome by using a larger sample volume in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results. 

Q14. Can the sensitivity of the kit assay be increased?

Yes. Samples with the lower concentrations of analyte will generate a lower absorbance change. For samples with low concentrations of analyte, a larger sample volume can be used in the assay to increase the absorbance change and thereby increase sensitivity of the assay. When doing this the increased volume of the sample should be subtracted from the distilled water volume that is added to the assay so that the total assay volume is unaltered. The increase sample volume should also be accounted for when calculating final results.

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Megazyme 琥珀酸检测试剂盒操作视频(K-SUCC)

Megazyme 溶解淀粉 操作视频

Megazyme 试剂盒样品前处理准备操作视频