UNIT 18.5 Detection of Phosphorylation by Enzymatic Techniques
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Shenolikar, S. 2001. Detection of Phosphorylation by Enzymatic Techniques. Current Protocols in Molecular Biology. 33:18.5:18.5.1–18.5.9.
- Published Online: 1 MAY 2001
- Published Print: JAN 1996
Reversible protein phosphorylation is an important mechanism for regulating physiological processes in both plant and animal cells. There are a number of techniques to demonstrate the presence of covalently bound phosphate in proteins. The general strategy of the protocols in this unit is to first examine the functional effects elicited by nonspecific acid or alkaline phosphatases that dephosphorylate many phosphoproteins in vitro. Protein phosphatases that selectively hydrolyze phosphoserine and phosphothreonine or phosphotyrosine residues can then be used to identify a functionally important covalent modification. Additional protocols describe digestion of phosphoproteins with a protein serine/threonine phosphatase and protein tyrosine phosphatase. A support protocol has been included to identify the radiolabel as 32Pi based on its ability to form a complex with ammonium molybdate.