Using bleach-chase to measure protein half-lives in living cells

被引：14

作者：

Geva-Zatorsky, Naama ^{[1
]}

Issaeva, Irina ^{[1
]}

Mayo, Avi ^{[1
]}

Cohen, Ariel ^{[1
]}

Dekel, Erez ^{[1
]}

Danon, Tamar ^{[1
]}

Cohen, Lydia ^{[1
]}

Liron, Yuvalal ^{[1
]}

Alon, Uri ^{[1
]}

Eden, Eran ^{[1
]}

机构：

[1] Weizmann Inst Sci, Dept Mol Cell Biol, IL-76100 Rehovot, Israel

来源：

NATURE PROTOCOLS | 2012年 / 7卷 / 04期

基金：

欧洲研究理事会;

关键词：

FLUORESCENT PROTEIN; DYNAMIC PROTEOMICS; GLOBAL ANALYSIS; UBIQUITIN; DEGRADATION; EXPRESSION; LIBRARY; PATHWAY; LOCALIZATION; STABILITY;

D O I：

10.1038/nprot.2012.028

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Protein removal has a central role in numerous cellular processes. Obtaining systematic measurements of multiple protein removal rates is necessary to understand the principles that govern these processes, but it is currently a major technical challenge. To address this, we developed 'bleach-chase', a noninvasive method for measuring the half-lives of multiple proteins at high temporal resolution in living cells. The method uses a library of annotated human reporter cell clones, each with a unique fluorescently tagged protein expressed from its native chromosomal location. In this protocol, we detail a simple procedure that bleaches the cells and uses time-lapse fluorescence microscopy and automated image analysis to systematically measure the half-life dynamics of multiple proteins. The duration of the protocol is 4-5 d. The method may be applicable to a wide range of fluorescently tagged proteins and cell lines.

引用

页码：801 / 811

页数：11

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