Abstract
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.
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Acknowledgements
We thank Z. Yakhini, Z. Kam, R. Milo, P. Tsvetkov, J. Adler and all members of the Alon laboratory for fruitful discussions and comments. We thank P. Choukroun for assistance with the computer cluster. This work was supported by the European Research Council, the Kahn Family Foundation and by Keren Isra-Pa'amei Tikva.
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E.E., N.G.-Z. and U.A. led the study and designed the experiments. E.E. and A.M. performed the analysis. N.G.-Z., T.D., L.C. and I.I. performed experiments. Y.L. designed the microscope operating software. E.D. and A.C. contributed to the analysis. E.E., N.G.-Z. and U.A. wrote the manuscript.
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Weizmann Institute of Science has filed for a patent on the technology described in this report for measuring protein half-lives.
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Geva-Zatorsky, N., Issaeva, I., Mayo, A. et al. Using bleach-chase to measure protein half-lives in living cells. Nat Protoc 7, 801–811 (2012). https://doi.org/10.1038/nprot.2012.028
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DOI: https://doi.org/10.1038/nprot.2012.028
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