TFIIA complex(Reconstitute from p55 and p12)

• Species: Human
• Expression Host: E.coli
• Tag: His-tag
• Purity: 90%
• Molecular Weight: 43.6 + 14.5 kDa.
• Gene Accession Number: NM_015859 +NM_004492.

Purification and Quality Control

p55 subunit of TFIIA is isolated form a strain of E. coli that contains the coding sequence of human p55 of TFIIA under the control of T7 promoter (3). p12 subunit of TFIIA is isolated from a strain of E. coli that contains the coding sequence for human TFIIA γ subunit under the control of T7 promoter. TFIIA was reconstituted as described.

Unit Definition (Activity)

1 unit equals 1 nanogram of purified protein. 1 unit is sufficient for a gel mobility shift assay in a 20 µl reaction; 100 units are sufficient for protein-protein interaction assays.

Applications

Recombinant TFIIA can be used to super-shift TBP-DNA complex in a gel mobility shift assay and to stimulate activator-dependent transcription in vitro (3, 5).

Formulation and Storage

The protein is in 20mM Tris-HCl pH7.9,100mM NaCl, 0.2mM EDTA, 1mM DTT and 20% glycerol. Stored at -70°C before use. Avoid repeated freeze thaw cycles.

Background

The transcription factor IIA (TFIIA) has been shown to bind to the TBP-DNA complex and to increase the affinity of TBP for the TATA element. Human TFIIA consists of three subunits of 35 kDa (α subunit), 19 kDa (β subunit) and 12 kDa (γ subunit). The α and β subunits are derived from the product, p55, of a single gene by an unknown mechanism. However, recombinant p55, in combination with a 12 kDa subunit (γ subunit), retains native TFIIA activity (1-4).

Image of SDS-PAGE / Western-blot