UNIT 18.5 Detection of Phosphorylation by Enzymatic Techniques

  1. Shirish Shenolikar

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb1805s33

Current Protocols in Molecular Biology

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.

Author Information

  1. Duke University Medical Center, Durham, North Carolina

Publication History

  1. Published Online: 1 MAY 2001
  2. 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.