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High‐Throughput, Single‐Step Purification of Affinity‐Tagged Protein Complexes
Author(s) -
LaCava John,
Hakhverdyan Zhanna,
Fenyo David,
Domanski Michal,
Oroskar Asha,
Oroskar Anil,
Hough Loren,
Jensen Torben Heick,
Chait Brian T.,
Rout Michael P.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.776.2
Subject(s) - protein purification , protein crystallization , affinity chromatography , protein–protein interaction , computational biology , microtiter plate , chromatography , protein precipitation , throughput , chemistry , target protein , tandem affinity purification , cell , extraction (chemistry) , computer science , biochemistry , biology , crystallization , enzyme , telecommunications , organic chemistry , wireless , gene
Most strategies for the capture of protein‐protein interactions by affinity chromatography still follow a trial and error approach to identify best conditions for interaction partner co‐precipitation. Moreover, in the case of some model systems (e.g. mammalian cell culture, animal tissue, etc.), the cost of obtaining enough material for extensive testing can be prohibitively expensive. Our lab has developed and successfully applied a high‐throughput, 96‐well protein affinity chromatography protocol to the yeast S. cerevisiae . We also demonstrate that this protocol is compatible with human cell culture. Our procedure requires a minimal amount of cell material per purification (~10–50 x less than typically used in a bench‐scale experiment), producing quantities of purified protein complexes that can be visualized by Coomassie blue stain even in the case of proteins of modest abundance (~1000 copies/cell). By testing 96 extraction conditions at a time we are able to greatly expand the number of known parameters affecting protein‐protein interactions. This approach increases the chances and reduces the time to find multiple instances of distinct protein complexes containing the tagged protein of interest, and should prove a useful tool for revealing protein‐protein interaction networks. The procedure is performed in a microtiter plate format, and so, is amenable to automation. NCRR ‐ 2U54 RR022220 ‐07