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 ([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.

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.