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Generation of a fluorescently labeled endogenous protein library in living human cells

Sigal, Alex and Danon, Tamar and Cohen, Ariel and Milo, Ron and Geva-Zatorsky, Naama and Lustig, Gila and Liron, Yuvalal and Alon, Uri and Perzov, Natalie (2007) Generation of a fluorescently labeled endogenous protein library in living human cells. Nature Protocols, 2 (6). pp. 1515-1527. ISSN 1754-2189. doi:10.1038/nprot.2007.197.

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We present a protocol to tag proteins expressed from their endogenous chromosomal locations in individual mammalian cells using central dogma tagging. The protocol can be used to build libraries of cell clones, each expressing one endogenous protein tagged with a fluorophore such as the yellow fluorescent protein. Each round of library generation produces 100–200 cell clones and takes about 1 month. The protocol integrates procedures for high-throughput single-cell cloning using flow cytometry, high-throughput cDNA generation and 3′ rapid amplification of cDNA ends, semi-automatic protein localization screening using fluorescent microscopy and freezing cells in 96-well format.

Item Type:Article
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Sigal, Alex0000-0001-8571-2004
Milo, Ron0000-0003-1641-2299
Additional Information:© 2007 Nature Publishing Group. Published online 14 June; corrected online 20 September 2007. We thank the Kahn Family Foundation and the Israel Science Foundation for support.
Funding AgencyGrant Number
Kahn Family FoundationUNSPECIFIED
Israel Science Foundation (ISF)UNSPECIFIED
Issue or Number:6
Record Number:CaltechAUTHORS:20150318-083942281
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:55874
Deposited By: Tony Diaz
Deposited On:18 Mar 2015 15:48
Last Modified:10 Nov 2021 20:51

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