Hai

I have'nt use any commercial kit,but i have folloewd the protocol given by SIGMA -(EC.2.7.1.2) it worksout for my study. Try it out.

Regards 

Rajakala

The most straightforward way is to use a coupled assay, where glucokinase converts glucose (at greater than minimally saturating concentration of glucose, 25mM or higher) to glucose-6-phosphate in the presence of ATP, which is then used as the substrate by the next enzyme glucose-6-phosphate dehydrogenase already present in the mixture along with other necessary components (NADP). NADP is quantitatively reduced to NADPH, kinetics of this reduction can be followed on a spectrophotometer and correlated to the activity of glucokinase, which is kept as the limiting variable. In the absence of a spectrophotometer, its possible to quantify the reduction reaction by a colorimetric set of reagents. I believe that many vendors sell these reagents in a kit form - for example Abcam. Be aware that to get accurate quantitation, like any other enzyme, you need the initial rate of reaction, corrected for proper controls.

According to Sigma Aldrich protocol, you can prepare samples using the following final assay concentrations: in a 3.00 ml reaction mix, the final concentrations are 60 mM Tris, 20 mM magnesium chloride, 4.0 mM adenosine  5'-triphosphate, 12.0 mM glucose, 0.9 mM ß-nicotinamide  adenine dinucleotide phosphate, 10 units glucose 6-phosphate dehydrogenase and 0.025 - 0.050 unit glucokinase. Monitor the  A340nm until constant using a suitably thermostatted  spectrophotometer. One unit will phosphorylate 1.0 µmole of D-glucose to D-glucose 6-phosphate per minute at pH 9.0 at 30°C.