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.
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Change history
20 September 2007
In the version of this article initially published, the authors omitted the following acknowledgment: “We thank the Kahn Family Foundation and the Israel Science Foundation for support.” The acknowledgment has been added to the PDF version of the article.
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Sigal, A., Danon, T., Cohen, A. et al. Generation of a fluorescently labeled endogenous protein library in living human cells. Nat Protoc 2, 1515–1527 (2007). https://doi.org/10.1038/nprot.2007.197
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DOI: https://doi.org/10.1038/nprot.2007.197
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