File:Strategy for affinity purification of Nanog associated protein complexes in mESCs..jpg
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DescriptionStrategy for affinity purification of Nanog associated protein complexes in mESCs..jpg |
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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[1] Direct
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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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current | 19:04, 5 April 2013 | 600 × 402 (77 KB) | Smallbot (talk | contribs) | Uploading CC-BY images from the the StemBook http://www.stembook.org/ 152/173 |
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