These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Purification of epitope-tagged transcription factor IID.
    Author: Carey MF, Peterson CL, Smale ST.
    Journal: Cold Spring Harb Protoc; 2010 Jul 01; 2010(7):pdb.prot5450. PubMed ID: 20647354.
    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 studies.
    [Abstract] [Full Text] [Related] [New Search]