Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield

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Pulse-Chase Labeling of Antigens with [35S]Methionine

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Performance of the pulse-chase method enables the investigator to determine the half-life (t1/2) of the protein of interest (Waterborg and Matthews 1984; L'Ecuyer et al. 1998; Huh and Wenthold 1999; Bonifacino 2001; Gromov and Celis 2001; Gerth et al. 2008; Chang et al. 2009). Upon translation, proteins become pulse-labeled by integration of [35S]methionine during their synthesis (the pulsed sample), and this is detected in an autoradiograph or by use of a phosphorimager. After a short labeling period of 15 min to 1 h in the presence of [35S]methionine, cells are cultured with an excess of unlabeled (cold) methionine (the chased sample), which competes with any remaining [35S]methionine for integration into newly translated proteins. Signal strength comparison of antigens in the pulsed sample with those in the chased samples allows for an estimation of the protein's relative half-life. The time interval for pulse labeling is dependent on the relative turnover of the antigen and may need to be optimized. Protein half-lives can vary from minutes to days; thus, the length of chase intervals may also need to be optimized. It should be noted that although most proteins decay at a rate that is independent of their concentration (zero-order kinetics), some initially display a quicker rate of decay followed by a longer rate, suggesting that multiple activities are affecting their half-life. All newly synthesized proteins will be labeled by this method; thus, an immunoprecipitation followed by protein separation using gel electrophoresis is required for detection of the specific target protein.

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

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