Unit

UNIT 18.8 Permeabilization Strategies to Study Protein Phosphorylation

  1. A. Nigel Carter

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb1808s40

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Carter, A. N. 2001. Permeabilization Strategies to Study Protein Phosphorylation. Current Protocols in Molecular Biology. 40:18.8:18.8.1–18.8.9.

Author Information

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

Publication History

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

Abstract

This unit deals with the use of nucleotide triphosphates to label proteins in vitro in permeabilized cells and isolated cellular fractions. These experiments generally utilize [g-32P]ATP as an exogenously added phosphate donor, although [g-32P]GTP can be used in specific cases. Procedures are outlined for performing a protein phosphorylation experiment using permeabilized cells and isolated intracellular organelles. Both of these procedures 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.