I am working in alpha glucosidase assay.....I prepared alpha glucosidase stock solution and kept in -10 degree C and when required diluted with buffer. But when apply PNPG as substrate the colour is not developing..Why it is happening? Note: I prepared fresh solution of PNPG
There are two possibilities: the enzyme is dead, or the conditions in which you are running the assay do not support enzyme activity.
Could you give more details about the way the enzyme was prepared and stored, and the buffer conditions of the enzyme assay? Are these methods similar to ones that have been used successfully by others for the same enzyme?
Have you tried varying the enzyme concentration? Have you tried preparing a fresh enzyme solution immediately before running the reaction?
Hi Tanmay
Just wondering what form of PNPG have you tested? make sure that its alpha and not beta, I don't know if the enzyme still active after been stored at -10? was it stored in proper solutions?
To exploit chromophoric priperties of p-nitrophenol, u need high pH. After termination of the assay, raise pH. U may see color
Dear Apurva Kumar when you talk about high pH, it is very vague can you please give a range?
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