Purification of epitope-tagged transcription factor IID.
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 studies.
Carey
Michael F
MF
Peterson
Craig L
CL
Smale
Stephen T
ST
D000906:Antibodies; D015153:Blotting, Western; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D005591:Chemical Fractionation; D002846:Chromatography, Affinity / Q000379:methods*; D000939:Epitopes / Q000378:metabolism*; D006367:HeLa Cells; D006388:Hemagglutinins; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D035362:Transcription Factor TFIID / Q000302:isolation & purification*
D000906:Antibodies; D002457:Cell Extracts; D000939:Epitopes; D006388:Hemagglutinins; D011993:Recombinant Fusion Proteins; D035362:Transcription Factor TFIID