File:Strategy for affinity purification of Nanog associated protein complexes in mESCs..jpg

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English: A. Establishment of a biotinylation system in ESCs. A stable ESC line expressing the bacterial BirA enzyme was first established by transfection with a BirA-expressing plasmid bearing the neomycin resistance (neor) gene and G418 selection; A second plasmid containing Nanog cDNA with an N-terminal Flag-biotin dual tag (FLBIO) and a puromycin resistance (puror) gene was introduced and cells selected with puromycin. The resulting stable lines are resistant to both G418 and puromycin and express FLAG-tagged, biotinylated Nanog that can be immunoprecipitated by anti-FLAG and streptavidin antibodies/beads. B. Two complementary affinity purification strategies for protein compexes purification. Single streptavidin immunoprecipitation and tandem affinity purification (anti-Flag immunoprecipitation followed by streptavidin pulldown) were performed in parallel, the purified protein complexes were fractionated on SDS-PAGE, and subjected to LC-MS/MS to identify components of the protein complexes.
Date Published July 14, 2008.
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StemBook Figure 4 Strategy for affinity purification of Nanog associated protein complexes in mESCs.

  • Wang, J., Trowbridge, J.J., Rao, S. and Orkin, S.H., Proteomic studies of stem cells (July 14, 2008), StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.4.1, http://www.stembook.org.
Author Wang, J., Trowbridge, J.J., Rao, S. and Orkin, S.H., Proteomic studies of stem cells (July 14, 2008), StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.4.1, http://www.stembook.org.
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