Purification of epitope-tagged transcription factor IID

Cold Spring Harbor Protocols
Michael F CareyStephen T Smale

Abstract

Transcription factor IID (TFIID) is one of the most critical factors in transcription complex assembly because it recognizes a core promoter and interacts with chromatin and activator proteins. This protocol uses immunoaffinity chromatography in a simple two-step procedure to purify modified TFIID to homogeneity with limited loss of activity. In brief, a short peptide containing the influenza virus hemagglutinin (HA) tag is fused onto the amino terminus of TATA-binding protein (TBP), and a retroviral transfer system is used to generate a HeLa cell line stably expressing the HA-tagged TBP. Extracts from this cell line contain TFIID, which stably incorporates the epitope-tagged TBP. The TFIID is partially purified from these extracts using phosphocellulose chromatography and then immunopurified using a resin containing protein A-Sepharose beads cross-linked to a monoclonal antibody against the influenza epitope. The TFIID is then eluted from the immunoaffinity resin in pure form using an HA peptide. The resulting TFIID contains a complete complement of TBP-associated factors (TAFs) and can be used in transcription, electrophoretic mobility shift assays (EMSA), and footprinting assays; its purity is well suited for many other stud...Continue Reading

References

Jan 1, 1983·Methods in Enzymology·J D DignamR G Roeder
Jan 1, 1996·Methods in Enzymology·E MaldonadoD Reinberg
Jan 1, 1996·Methods in Enzymology·D TantinM Carey
Feb 13, 2010·Cold Spring Harbor Protocols·Michael F CareyStephen T Smale
Jan 1, 2006·CSH Protocols·Richard J Simpson
Jan 1, 2006·CSH Protocols·Ed Harlow, David Lane

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Citations

Dec 18, 2013·Biotechnology Advances·Ana Sofia PinaAna Cecília A Roque
Nov 3, 2010·Cold Spring Harbor Protocols·Michael F CareyStephen T Smale

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