UNIT 18.8 Permeabilization Strategies to Study Protein Phosphorylation
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 MAY 2001
- Published Print: OCT 1997
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.