Abstract
p53, is known as the guardian of the genome and as such requires exquisite regulation not only of its abundance but also its activity. The abundance of p53 can be modulated at the level of transcription, translation, and also via its degradation.This protocol involves 35S metabolic labelling of newly synthesized proteins followed by a period of chase with "cold" media. Samples are harvested and p53 immunopreciptiated, separated by SDS PAGE and the levels of 35S labelled p53 determined. By comparing the level of 35S p53 at 0 h to those "chased" with cold media (e.g. 60 min) provides an indication of the rate of p53 turnover.
Materials and Reagents
Equipment
Software
Procedure
Recipes
Acknowledgments
This protocol was originally published in its brief version in Astle et al. (2012). This work was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia to RDH (NHMRC Nos. 166908 and 251688) and to RBP (NHMRC Nos. 509087 and 400116) and from Cancer Council Victoria to RBP. RDH and RBP are NHMRC Senior Research Fellows. We acknowledge the Victorian Centre for Functional Genomics for the use of equipment and advice. We thank Professor Stephen Jane and Loretta Cerruti for assistance with retrovirus production.
References
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