title: Using bleach-chase to measure protein half-lives in living cells.
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.
authors.affiliation: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Naama
authors.initials: N
authors.lastname: Geva-Zatorsky
authors.forename: Irina
authors.initials: I
authors.lastname: Issaeva
authors.forename: Avi
authors.initials: A
authors.lastname: Mayo
authors.forename: Ariel
authors.initials: A
authors.lastname: Cohen
authors.forename: Erez
authors.initials: E
authors.lastname: Dekel
authors.forename: Tamar
authors.initials: T
authors.lastname: Danon
authors.forename: Lydia
authors.initials: L
authors.lastname: Cohen
authors.forename: Yuvalal
authors.initials: Y
authors.lastname: Liron
authors.forename: Uri
authors.initials: U
authors.lastname: Alon
authors.forename: Eran
authors.initials: E
authors.lastname: Eden
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis; D057886:Bleaching Agents / Q000494:pharmacology; D017930:Genes, Reporter; D006207:Half-Life; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D059748:Proteolysis; D059008:Time-Lapse Imaging
chemical_list: D001426:Bacterial Proteins; D057886:Bleaching Agents; D008164:Luminescent Proteins; D011506:Proteins; C054562:yellow fluorescent protein, Bacteria