I'm currently doing the assay for inhibition of glucosidase.
50 uL of extract and 50 uL of glucosidase were incubated for 15 mins at 37 C. Then, 50 uL of 4-nitrophenyl-a-D-glucopyranoside. It was incubated for 30 mins at 37 C. 0.1 M Na2CO3 were added to stop the reaction. No yellow formation is produced unless I boil the mixture. When I boil the mixture, even the blank (having no enzyme) turns into yellow.
How to solve the problem?
Hi there,
The optimistic would say that the pretreatment of the glucosidase with the extract results in the full inhibition of the enzyme. The pessimistic would say the extract might degrade the enzyme or the enzyme is simply inactive from the start. Did you run the control assay where the extract is replaced by buffer? It should normally result in the full activity of the enzyme (ie. yellow appearing when adding Na2CO3).
Dominique Liger
I also did the control assay (100 percent enzyme activity). Same thing happens. No yellow formation upon addition of Na2CO3. The yellow color only appears if you boil the mixture.
Well, if the control is not working at all there is something wrong with the enzyme and/or with the assay...
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