Unit

UNIT 13.8 Permeabilization Strategies to Study Protein Phosphorylation

  1. A. Nigel Carter

Published Online: 1 MAY 2001

DOI: 10.1002/0471140864.ps1308s10

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Carter, A. N. 2001. Permeabilization Strategies to Study Protein Phosphorylation. Current Protocols in Protein Science. 10:13.8:13.8.1–13.8.19.

Author Information

  1. The Salk Institute for Biological Studies, La Jolla, California

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: DEC 1997

Abstract

This unit deals with the use of nucleotide triphosphates to label proteins in vitro in permeabilized cells and isolated cellular fractions. Both of these assay formats result in lysates from which the protein of interest may be easily immunoprecipitated; however alternative techniques are described for preparing the final lysate for electrophoretic analysis. A related procedure that does not involve permeabilization is outlined for direct analysis of cytosolic or membrane-bound kinases. Two different methods for determining the specific radioactivity of 32P-containing compounds are also included. These experiments generally utilize [γ-32P]ATP as an exogenously added phosphate donor, although[γ-32P]GTP can be used in specific cases. The method is very straightforward, although numerous considerations must be made before applying it to each new system.