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

Alex Sigal, Tamar Danon, Ariel Cohen, Ron Milo, Naama Geva-Zatorsky, Gila Lustig, Yuvalal Liron, Uri Alon, Natalie Perzov

Research output: Contribution to journalArticlepeer-review

52 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)1515-1527
Number of pages13
JournalNature Protocols
Volume2
Issue number6
DOIs
StatePublished - 1 Jun 2007
Externally publishedYes

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