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

Nature Protocols volume 2, pages 1515–1527 (2007)Cite this article

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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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Figure 1: Generation of a CD-tagged protein library.
Figure 2: Flowchart of the library generation procedure.
Figure 3: Optical configurations and stream alignments for YFP- and mCherry-based cell sorting.
Figure 4: Cell sorting of positive clones by flow cytometry.
Figure 5: Characterization of tagged cell clones.
Figure 6: Timescale of library construction.

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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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Author notes

  1. Alex Sigal

    Present address: Present address: Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.,

Authors and Affiliations

  1. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 76100, Israel

    Alex Sigal, Tamar Danon, Ariel Cohen, Naama Geva-Zatorsky, Yuvalal Liron, Uri Alon & Natalie Perzov

  2. Department of Systems Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Ron Milo

  3. Department of Biological Regulation, Weizmann Institute of Science, Rehovot, 76100, Israel

    Gila Lustig

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Correspondence to Alex Sigal.

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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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