I am working on an alpha-glucosidase assay. But when I apply PNPG as the substrate, the color is not developing. Why is it happening? Note: I prepared a fresh solution of PNPG and prepared the fresh enzyme solution immediately before running the reaction.
3/30/23
Dear Nimashi,
What kind of assay are you doing for the a-glucosidase? Is it a fixed-time or kinetic assay? What conditions are you using when you test the enzyme, e.g., what buffer, pH, temperature, etc. are you using? How do you stop the reaction? The enzyme cleaves PNPG to release para-nitro-phenol (PNP) which is yellow. In reality, PNP itself is not yellow, but it ionizes to form the p-nitrophenolate anion. The anion (not protonated PNP) is yellow. To visualize the yellow color, the pH has to be >5.9. If you are using a buffer at a pH
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