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 ([email protected]). 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.