The His-tag recombinant protein is purified by affinity chromatography in combination with FPLC columns. The purified RNA pol II-hRPB3 is greater than 70% homogeneous based on SDS-PAGE analysis.
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; 20 units are sufficient for in vitro transcription assay and 100 units are sufficient for protein-protein interaction assays.
Applications
RPB3 has been applied in protein-protein interactions assays..
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.
In human, RPB3 is encoded by the POLR2C
gene (1). This gene encodes the third largest subunit of RNA polymerase II. The product of this gene contains a cysteine rich region and exists as a heterodimer with another polymerase subunit, POLR2J. These two subunits form a core subassembly unit of the polymerase. DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. Pol II is the central component of the basal RNA polymerase II transcription machinery. RPB3 is part of the core element with the central large cleft and the clamp element that moves to open and close the cleft. In the pol II complex, RPB3 interacts with RPB5 and the RPB3–RPB5 interaction is intensified in the presence of three subunits, RPB7, RPB8 and RPB11. Besides,RPB3 also interacts with RPB1-5, 7, 10-12 (2).
RPB3 is involved in tissue-specific transcription and muscle differentiation via interaction with the myogenic factor Myogenin (3). RPB3 also interact with IGFBP3 (4).
