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

Summary: Generation of a fluorescently labeled endogenous
protein library in living human cells
Alex Sigal1,4, Tamar Danon1, Ariel Cohen1, Ron Milo2, Naama Geva-Zatorsky1, Gila Lustig3, Yuvalal Liron1,
Uri Alon1 & Natalie Perzov1
1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel. 2Department of Systems Biology, Harvard Medical School, Boston,
Massachusetts 02115, USA. 3Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel. 4Present address: Division of Biology, California
Institute of Technology, Pasadena, California 91125, USA. Correspondence should be addressed to A.S. (sigal@caltech.edu).
Published online 14 June; corrected online 20 September 2007 (details online); doi:10.1038/nprot.2007.197
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 100200 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.
INTRODUCTION
A quantitative understanding of human protein networks requires
the measurement of endogenous protein dynamics in living cells1.
An ideal measurement system would: (a) work at the protein level,
because the regulation of translation, localization and degradation
is crucial in mammalian cells, (b) work at the level of individual

  

Source: Alon, Uri - Departments of Molecular Cell Biology & Physics of Complex Systems, Weizmann Institute of Science

 

Collections: Biology and Medicine