Electrophoretic separation of protein kinases: altered mobility with different crosslinking agents in the presence of certain detergents.
Nondenaturing polyacrylamide gel electrophoresis was used to separate protein kinases in crude extracts and subcellular fractions of murine erythroleukemic cells. The kinases were detected using an in situ phosphorylation assay. The electrophoretic patterns obtained using gel bound to GelBond and prepared with AcrylAide differed from those seen without GelBond and with N,N'-methylenebisacrylamide as cross-linker. In an attempt to improve the resolution of the bands in the membrane fractions, detergent-treated preparations were electrophoresed through gels which contained either 0.1% Triton X-100 or 0.1% Nonidet P-40. The resolution of the bands in this fraction was not, however, improved with the inclusion of the nonionic detergent in the gels. When cytosol was electrophoresed through gels containing detergent, a major band of cAMP-dependent protein kinase activity showed a marked shift in mobility. This may have been the result of a structural change, altering the shape and possibly affecting the charge on the molecule, or the enzyme may have formed aggregates with the detergent.
Sprott
S
S
Department of Medical Biochemistry, University of the Witwatersrand Medical School, Parktown, Johannesburg, South Africa.
Hammond
K D
KD
Savage
N
N
D000081:Acetamides / Q000494:pharmacology; D000178:Acrylamides; D000818:Animals; D003432:Cross-Linking Reagents; D000242:Cyclic AMP / Q000494:pharmacology; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011494:Protein Kinases / Q000302:isolation & purification*; D013501:Surface-Active Agents; D014407:Tumor Cells, Cultured
D000081:Acetamides; D000178:Acrylamides; D003432:Cross-Linking Reagents; D003902:Detergents; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D000242:Cyclic AMP; D011494:Protein Kinases; C021221:N,N'-methylenebisacrylamide; C014026:hexamethylene bisacetamide