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.

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.

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.

The kit assay will only measure the non-covalently linked monosaccharide.

Oligosaccharides or polysaccharides can be measured after hydrolysis to the monosaccharide. Generally acid hydrolysis can be achieved by boiling the oligo/polysaccharide in 1.3 M HCl for 1 h. It is recommended that scientific literature is consulted for information on hydrolysis conditions for the particular oligo/polysaccharide that is being measured.

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.

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.

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.

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).

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.

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.

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.

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.

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. 

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.

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.

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.

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.

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.

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.

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.

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).

The kit assay will only measure the non-covalently linked monosaccharide.

Oligosaccharides or polysaccharides can be measured after hydrolysis to the monosaccharide. Generally acid hydrolysis can be achieved by boiling the oligo/polysaccharide in 1.3 M HCl for 1 h. It is recommended that scientific literature is consulted for information on hydrolysis conditions for the particular oligo/polysaccharide that is being measured.

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.

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.

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.

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.

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.

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.

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. 

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.

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.

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.

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.

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.

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).

The kit assay will only measure the non-covalently linked monosaccharide.

Oligosaccharides or polysaccharides can be measured after hydrolysis to the monosaccharide. Generally acid hydrolysis can be achieved by boiling the oligo/polysaccharide in 1.3 M HCl for 1 h. It is recommended that scientific literature is consulted for information on hydrolysis conditions for the particular oligo/polysaccharide that is being measured.

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.

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:
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.
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).

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.

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.

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.

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.

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.

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.

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.

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. 

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.

Yes.  This kit can be used to rapidly and simply quantify D-gluconic acid in soy sauce, with only the following slight modification to the analysis procedure as described in the booklet:
(a) Sample preparation:
Take 2 mL of soy sauce, add 4 mL of Carrez I solution (see page 7 of booklet) and mix. Then add 4 mL of Carrez II solution (see page 7 of booklet) and mix thoroughly.  Filter the resultant suspension through Whatman No. 1 filter paper without any further additions (in this case, sodium hydroxide is NOT added to neutralise the extract).  The filtrate should be clear and may be slightly yellow / orange in colour (this is normal).
(b) Assay procedure:
Use up to 0.5 mL of the filtrate according to the normal assay procedure (see page 5 of booklet) – however, after the addition of 0.02 mL of suspension 3 (6-PGDH), the cuvettes (after mixing) should be incubated for 10 min (e.g. on the bench top or in the spectrophotometer compartment) prior to recording the A1 values.  This is to allow colour complexes time to stabilise, thus enabling the rise in absorbance due to the conversion of D-gluconic acid to be measured accurately.  The speed of the reaction on addition of the gluconate kinase is unaffected by the sample preparation / sample size, and will take just a few minutes to complete as normal.

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.

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.

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.

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. 

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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. 

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.

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.

The test is best run at pH 7.4.  The reagent is buffered at this pH.  Using different pH values will affect results, i.e. it may take longer to reach this end point; but the same end-point value should be obtained if not too far away from this pH value.

The test is set up to measure between 10 and 100 micrograms per assay (i.e. 0.1 mL of 0.1 to 1.0 mg/mL).  You may prefer to use 3.0 mL of GOPOD reagent mixture plus 1.0 mL of sample; in this case the concentration range in the material being analysed (diluted) should be ~10 to 100 micrograms per mL.  The test is linear up to an absorbance of 1.4 (final assay volume of 3.2 mL).  If the final volume is 4.0 mL, then linearity will be up to about 1.0 absorbance units.

The reaction is complete after approx. 15 min, and the colour is stable at temperatures of 20-50˚C for at least an extra hour.

1 microgram gives an absorbance of 0.01 if 3 mL of GOPOD are used.  If 1 mL of GOPOD is used, 1 microgram gives an absorbance of 0.03 OD. 

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.

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.

The kit enzymes can be stored at 0-5°C for up to 12 months, so they will be perfectly fine.

The temperature is just for the incubations.  It is an end-point assay, so the spectrophotometer does not need to be temperature controlled.

The kit assay will only measure the non-covalently linked monosaccharide.

Oligosaccharides or polysaccharides can be measured after hydrolysis to the monosaccharide. Generally acid hydrolysis can be achieved by boiling the oligo/polysaccharide in 1.3 M HCl for 1 h. It is recommended that scientific literature is consulted for information on hydrolysis conditions for the particular oligo/polysaccharide that is being measured.

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.

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.

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.

No.  The K-GLUHKL test kit is specific for the measurement of “free” D-glucose.  It will not detect glucose that is bound by a glycosidic linkage to another sugar molecule. 

Yes.  It is possible that biological samples may be used directly after appropriate sample dilution in distilled water, however some biological samples may require deproteinisation with perchloric acid prior to addition to the assay.  The deproteinisation method can be found at the following link on the Megazyme website: Deproteinisation Method

Dilution during sample preparation must be taken into account in the final calculation.

Yes.  Determination of D-glucose in polysaccharides and fibrous plant material: 
Mill plant material or polysaccharide to pass a 0.5 mm screen using a Retsch centrifugal mill, or similar.  Accurately weigh approx. 100 mg of material into a Corning screw-cap culture tube (16 x 125 mm).  Add 5 mL of 1.3 M HCl to each tube and cap the tubes.  Incubate the tubes at 100˚C for 1 h.  Stir the tubes intermittently during the incubation.  Cool the tubes to room temperature, carefully loosen the caps and add 5 mL of 1.3 M NaOH.  Quantitatively transfer the contents of the tube to a 100 mL volumetric flask using distilled water and adjust the volume to 100 mL with distilled water.  Mix thoroughly by inversion and filter an aliquot of the solution through Whatman No. 1 filter paper or centrifuge at 1,500 g for 10 min.  Typically, no further dilution is required and a sample volume of 0.1 mL is satisfactory. 

The kit assay will only measure the non-covalently linked monosaccharide.

Oligosaccharides or polysaccharides can be measured after hydrolysis to the monosaccharide. Generally acid hydrolysis can be achieved by boiling the oligo/polysaccharide in 1.3 M HCl for 1 h. It is recommended that scientific literature is consulted for information on hydrolysis conditions for the particular oligo/polysaccharide that is being measured.

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.

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.

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.

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.

No.  The K-GLUHKL test kit is specific for the measurement of “free” D-glucose.  It will not detect glucose that is bound by a glycosidic linkage to another sugar molecule. 

Yes.  It is possible that biological samples may be used directly after appropriate sample dilution in distilled water, however some biological samples may require deproteinisation with perchloric acid prior to addition to the assay.  The deproteinisation method can be found at the following link on the Megazyme website: Deproteinisation Method

Dilution during sample preparation must be taken into account in the final calculation.

Yes.  Determination of D-glucose in polysaccharides and fibrous plant material: 
Mill plant material or polysaccharide to pass a 0.5 mm screen using a Retsch centrifugal mill, or similar.  Accurately weigh approx. 100 mg of material into a Corning screw-cap culture tube (16 x 125 mm).  Add 5 mL of 1.3 M HCl to each tube and cap the tubes.  Incubate the tubes at 100˚C for 1 h.  Stir the tubes intermittently during the incubation.  Cool the tubes to room temperature, carefully loosen the caps and add 5 mL of 1.3 M NaOH.  Quantitatively transfer the contents of the tube to a 100 mL volumetric flask using distilled water and adjust the volume to 100 mL with distilled water.  Mix thoroughly by inversion and filter an aliquot of the solution through Whatman No. 1 filter paper or centrifuge at 1,500 g for 10 min.  Typically, no further dilution is required and a sample volume of 0.1 mL is satisfactory. 

The kit assay will only measure the non-covalently linked monosaccharide.

Oligosaccharides or polysaccharides can be measured after hydrolysis to the monosaccharide. Generally acid hydrolysis can be achieved by boiling the oligo/polysaccharide in 1.3 M HCl for 1 h. It is recommended that scientific literature is consulted for information on hydrolysis conditions for the particular oligo/polysaccharide that is being measured.

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.

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.

Procedure B is required  for  “low-lactose” or “lactose-free” samples containing high levels of monosaccharides.  Generally, these types of samples contain high levels of  “free” galactose which causes a high background and reduces the dynamic range available to measure the galactose that is released from lactose in the test.  To avoid this the borohydride step is used to reduce the free galactose.

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.

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.

Instead of the normal Carrez sample treatment, 1 mL of milk is added to 4 mL of water and 1 mL of 10 mg/mL sodium borohydride (dissolved in 50 mM NaOH and less than 5 hours old).  This solution is incubated in a closed plastic container at 40˚C for 30 min, after which it is neutralised by the addition of 2.5 mL of 0.2 M acetic acid, and then simply filtered through Whatman No. 1 filter paper.  The extract, that will be hazy, is analysed without any further treatment, and 0.2 mL per assay should be used (according to the normal procedure).  Although the samples are all hazy, this haze is stable in the assay and contributes very little to the absorbance.
In the assay the borohydride reduces all reducing sugars in the milk to their sugar alcohols, i.e. glucose goes to sorbitol, galactose goes to galactitol, and the residual lactose that we are interested in goes to lactitol.  Then the usual beta-galactosidase in the kit hydrolyses the produced lactitol into galactose and sorbitol.  However, as the borohydride has been neutralised at this stage, the galactose remains as galactose, and thus can be acted upon and quantified by the galactose dehydrogenase.
The extra reagents (to K-LACGAR) that are required to perform such analyses are:

1. Sodium borohydride (Sigma S-9125)
2. 50 mM NaOH
3. 0.2 M acetic acid

Note: After borohydride reduction of the sample the incubation step with beta-galactosidase should be increased to 1 hour.

Yes, this is possible. Here are two options for sample preparation methods for pastry products:
1. Mill or homogenise sample materials.  Weigh out a representative sample and extract with water (heated to 60˚C if necessary).  Quantitatively transfer to a volumetric flask and dilute to the mark with distilled water.  Mix, filter and use the appropriately diluted, clear solution for the assay.
Alternatively the sample can be treated with Carrez reagents after the extraction with water:
2. Mill or homogenise sample materials.  Accurately weigh approx. 1 g of into a 100 mL volumetric flask, add approx. 40 mL of distilled water, mix and store at 60˚C for 15 min with occasional swirling.  Add 2 mL of Carrez II solution and mix.  Add 2 mL of Carrez I solution and mix.  Add 4 mL of 100 mM NaOH solution and mix vigorously. Dilute to volume with distilled water and mix thoroughly.  Filter an aliquot of the solution through Whatman No. 1 filter paper.
Discard the first few mL of filtrate.  Use the clear filtrate (sample solution) in the assay.  Alternatively centrifuge in a microfuge tube at 13000 x rpm and using the clear supernatant in the assay.
The procedures given here can be modified to suit the sample, e.g. the dilution effect of the lactose in the sample can be reduced by extracting the sample in a lower volume of water so that the final concentration of lactose is detectable by the kit.  This would need to be assessed by the user.

The linear range of 0.008 – 0.16 g/L is based on the recommended minimum absorbance change of 0.1, however some users are comfortable working below this level hence the limit of detection is based on a 1 mL sample volume and minimum absorbance change of 0.02.  The sample volume can be altered; for samples containing low concentrations of lactose the sample volume can be increased to up to 1 mL however the distilled water volume must be altered accordingly so that the final assay volume is not altered, otherwise the calculation will be affected (see page 7 of the K-LACGAR booklet).  For concentrated samples these should be diluted in distilled water and the dilution factor included into the calculation.

It is possible to measure the K-LACGAR reactions at 365 nm.  In this instance the extinction coefficient of NADH alters from 6300 [L x mol-1 x cm-1] to 3400 [L x mol-1 x cm-1] and this must be accounted for in the calculation of D-glactose and lactose.  The calculations for measurements recorded at 365 nm are shown below.

Note: Alternatively the MegaCalc application may be used for easy processing of raw data values, however if the MegaCalc application is used for calculations recorded at 365 nm then the calculated values (g/L) must be multiplied by 1.8529.

This kit can be used as described in the format below to measure L-arabinose but not D-arabinose:
FORMAT:
Wavelength:                340 nm
Cuvette:                       1 cm light path (glass or plastic)
Temperature:              ~ 25°C
Final Volume:              2.72 mL
Sample solution:         4 – 120 μg of L-arabinose per cuvette
Read against air:        without cuvette in light path

* for example with a plastic spatula or by gentle inversion after closing the cuvette with a cuvette cap or Parafilm®.
** if this “creep” rate is greater for the sample than for the blank, extrapolate the absorbances (sample and blank) back to the time of addition of suspension 5.
CALCULATION:
Determine the absorbance difference (A2-A1) for both blank and sample.  Subtract the absorbance difference of the blank from the absorbance difference of the corresponding sample, thereby obtaining DA.
The concentration of arabinose can be calculated as follows:

where:
V     = final volume [mL]
MW = molecular weight of arabinose [g/mol]
ε      = extinction coefficient of NAD+ at 340 nm
= 6300 [L x mol-1 x cm-1]
d      = light path [cm]
v      = sample volume [mL]

If the sample has been diluted in addition to the dilution during preparation, the result must also be multiplied by the additional dilution factor, F.
When analysing solid and semi-solid samples which are weighed out for sample preparation, the content (g/100 g) is calculated from the amount weighed as follows:

As a general rule: Procedure A is used for samples that are known to contain low levels of free galactose. Procedure B is used for samples that are known to contain high levels of free galactose. If the free galactose content of a sample is unknown it is recommend that the galactose is measured as per the galactose assays in Procedure A.
For “lactose free” samples it is generally recommended that procedure B is used to maximise the absorbance range and enable the sensitive detection of lactose.
“Lactose free” dairy products have usually been processed whereby lactose in the original sample has been hydrolysed to glucose and galactose. These samples will contain high levels of free galactose and should be processed using procedure B. Some samples will be “lactose free” because the original sample never contained lactose so, assuming that the free galactose level is low, these samples can be processed using procedure A.

Step 1: Add 1 g of sample (or homogenised sample) to 4 mL of water, mix then add 1 mL of 10 mg/mL sodium borohydride (dissolved in 50 mM NaOH and less than 5 hours old).  Incubate this solution in a sealed plastic container at 40°C for 30 min then neutralise by the addition of 2.5 mL of 0.2 M acetic acid.  Transfer all of the borohydride reduced sample (~ 8.5 mL) to a 10 mL volumetric flask and make the final volume to 10 mL with distilled water. Filter through Whatman No. 1 filter paper or centrifuge in a microfuge at 13000 x g and use the filtrate or supernatant directly in the assay or with an appropriate dilution in distilled water (if required).  The filtrate may be hazy but this is stable in the assay and contributes very little to the absorbance.  Typically use a sample volume of 0.2 mL in Step 2 of PROCEDURE B.
For analysis of results the dilution is 1 and the concentration of the prepared sample is 100 g/L (i.e. 1 g prepared in 10 mL).

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.

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).

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.

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.

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.

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.

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.

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.

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. 

The K-LACSU kit will allow measurement of lactose in any materials.

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.

The Lactose/Sucrose/D-Glucose Kit should work fine for wort.

Yes, it is possible to specifically measure lactose in a mixture of sucrose, lactose and glucose.  Of course, it is also necessary to measure free glucose (i.e. with no added beta-galactosidase).

The Lactose/Sucrose/D-Glucose Kit will allow measurement of sucrose, lactose and  glucose (not total sugars).  We can supply reagents separately for sucrose or lactose determination (or Glucose).

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.

The kits we supply are stable at room temperature for several months.  Thus we ship at room temperature.  When you get the kit, it should be stored as recommended and it will be stable for several years.  The kit will be perfectly fine at 30˚C for the shipping time.

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.

Samples that generate absorbance values A₂ – A₁ of 0.3 should be diluted in distilled water prior to the Sample Preparation (section A, page 7) and the second incubation of step 2 increased (glucose oxidase / catalase) to 30 min. 

Some critical steps of the assay are as follows:
A₂ should be read after approximately 10 min and you should ensure that the reaction has finished, i.e. measure the absorbance until it stops increasing.  (Slight increases in absorbance of 0.001/min or less are acceptable).
The supernatants from both steps (1 and 2) of A. Sample Preparation should be clear. 

The K-LACTUL kit will measure lactulose in most samples however it is the sample preparation prior to the Enzymatic Determination Reaction that is important. Megazyme has only tested milk-based samples, however most samples that do not contain high protein levels may work using the same standard procedure as described in the K-LACTUL data booklet. Samples containing very high levels of free fructose may not work.

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.

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.

If it is suspected that the measurements of K-LACTUL are not correct and there is doubt regarding the performance of the kit then the following steps should be checked.
1. Check that the cuvettes are 1.5 mL microcuvettes and that the volume of the liquid in the cuvettes is high enough for the spectrophotometer.
2. Check the temperature of the reactions is correct.
Using the standard lactulose/fructose solution (bottle 8) that is supplied with the kit will help determine where issues are occurring with the measurement of lactulose samples.  The obvious steps where issues may occur are: A. Sample Preparation (page 7 K-LACTUL booklet) and B. Enzymatic Determination Reaction (page 8 K-LACTUL booklet).
3. The performance of K-LACTUL can be tested as follows:
(A. Sample Preparation (page 7 K-LACTUL booklet)
Use 0.5 mL of the standard lactulose /fructose solution (Bottle 8) which contains 0.1 mg/mL lactulose and 0.05 mg/mL fructose.  The typical individual absorbance values are: A1 = 0.2, A2 = 0.2, A3 = 1.0.  This should generate a final absorbance difference of (A3-A2) of approximately 0.8 (Note: this measurement includes the lactulose and fructose measurement and is not just lactulose content only).
Note: If the correct values are obtained for the performance of K-LACTUL then there is no need to check the performance of the Enzymatic Determination Reaction step separately.
4. The performance of the Enzymatic Determination Reaction step can be tested separately as follows:
B. Enzymatic Determination Reaction (page 8 K-LACTUL booklet)
This test uses 0.1 mL of the standard lactulose /fructose solution (Bottle 8) which contains 0.05 mg/mL fructose. This is equivalent to 5 μg of fructose added to the cuvette and should generate an absorbance difference (A3-A2) of approximately 0.3. If this absorbance difference is obtained then it can be concluded that the step is performing correctly.
B. ENZYMATIC DETERMINATION REACTION:
Wavelength: 340 nm
Cuvette: 1 cm light path (glass or plastic; 1.5 mL semi-micro)
Final volume: 1.16 mL
Sample solution: 0.65-65 μg of lactulose per cuvette (in 0.1-1.0 mL sample volume)
Read against air (without cuvette in the light path) or against water Pipette

* for example with a plastic spatula or by gentle inversion after sealing the cuvette with a cuvette cap or Parafilm®. 

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.

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).

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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. 

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.

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. 

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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

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.

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.

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.

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. 

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. 

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. 

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.

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.

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).

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.

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.

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

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.

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.

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.

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-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. 

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

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. 

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 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.

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. 

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.

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.

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

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.

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).

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.

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.

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.

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.

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

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.

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-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. 

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. 

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 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.

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. 

Propionate may react more slowly than acetate.

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.

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.

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.

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).

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.

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.

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 therefor

Propionate may react more slowly than acetate.

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. 

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.

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.

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

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. 

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.

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-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. 

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 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.

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. 

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.

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.

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.

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).

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.

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.

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)

Preparation of R2: (90 assays)

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) 

Preparation of R2: (90 assays)

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

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.

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.

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.

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.

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.

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. 

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.