The turnover rate of different protein species in a signal transduction network strongly affects the impact of the given species on the outcome of a stimulus. Whereas stable, long-lived proteins mainly account for the transmission of a signal, unstable short-lived species often comprise regulatory functions. Here, we describe a method to determine the half-lives of proteins of the JAK/STAT pathway by a pulse-chase approach in cell culture. First, radioactive labeling with 35S-methionine is carried out to label newly synthesized proteins (pulse). Subsequently, the dynamics of the decay of these proteins is monitored in the absence of labeled amino acids over a de fi ned time period (chase). For this purpose the protein of interest is isolated by immunoprecipitation from total cell lysates, separated on an SDS-polyacrylamide gel, and subsequently visualized by autoradiography. © Springer Science+Business Media New York 2013.
CITATION STYLE
Dittrich, A., Siewert, E., & Schaper, F. (2013). Determination of protein turnover rates in the JAK/STAT pathway using a radioactive pulse-chase approach. Methods in Molecular Biology, 967, 69–80. https://doi.org/10.1007/978-1-62703-242-1_5
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