Protein Kinase C Protocols

Volume 233 of the series Methods in Molecular Biology™ pp 163-170

Pulse-Chase Analysis of Protein Kinase C

  • Mikiko TakahashiAffiliated withBiosignal Research Center, Kobe University
  • , Yoshitaka OnoAffiliated withBiosignal Research Center, Kobe University

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Pulse-chase experiments allow study of the fate of proteins after synthesis, such as processing, intracellular transport, secretion, degradation, and physical chemical properties of proteins. Pulse-chase protocols have been used to analyze the phosphorylation of endogenous and transiently expressed protein kinase C (PKC) family proteins (15). To radiolabel newly synthesized proteins, the cells are incubated with radiolabeled amino acid for short periods (pulse labeling). Then the pulse is followed by a chase in which cells are further incubated with the excess amount of unlabeled counterpart of the precursor used for labeling. The radiolabeled protein of interest is analyzed by electrophoresis after isolation from other cellular proteins by immunoprecipitation.