Alkaline phosphatase - (Sep/05/2001 )
I read in the Dako Immunochemical Staining Methods Handbook that phosphate buffers should not be used in procedures involving alkaline phosphatase, as activity of the enzyme will be inhibited. Has anyone found this to be true? If yes, what would you recommend as a standard buffer for ELISA assays?
It is true, but irrelevant. Phosphatases are (surprise-surprise) indeed inhibited by phosphate buffer, but as long as the substrate buffer is NOT a phosphate buffer, you have no problem. Many people use a Tris/Tween buffer for general ELISA. Works for both HRP and ALK-PHOS.
My lab uses diethanolamine buffer 0.075 M pH 9.3 for alkaline phosphatase assays...it is true that that phosphate is a product inhibitor of the enzyme