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Author:
Date:
2012-11-05
[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.
[摘要] p53，被称为基因组的守护者，因此需要精细的调节不仅其丰度，而且其活性。 p53的丰度可以在转录，翻译以及通过其降解的水平上调节。
该方案涉及新合成的蛋白质的35S代谢标记，随后用"冷"培养基追踪一段时间 。 收获样品，并通过SDS PAGE分离p53免疫沉淀，并测定35S标记的p53的水平。 通过将0h时35S p53的水平与用冷培养基（例如60分钟）的"追踪"水平进行比较提供了p53周转速率的指示。
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