title: Permeabilization strategies to study protein phosphorylation.
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-(32)P]ATP as an exogenously added phosphate donor, although [g-(32)P]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 (32)P -containing compounds are also included.
authors.affiliation: The Salk Institute for Biological Studies, La Jolla, California, USA.
authors.forename: A N
authors.initials: AN
authors.lastname: Carter
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D002463:Cell Membrane Permeability; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D047468:Immunoprecipitation; D007525:Isoelectric Focusing; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D013347:Subcellular Fractions
chemical_list: D010761:Phosphorus Radioisotopes; D011506:Proteins; D000255:Adenosine Triphosphate