Monoclonal antibody raised in mouse against the YSPTSPS repeat in the B1 subunit of RNA polymerase II
Protein A purified monoclonal antibody in PBS containing 0.05% azide.
Aqueous buffered solution containing 1% BSA, 50% glycerol and 0.09% sodium azide.
Store at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles
DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. Largest and catalytic component of RNA polymerase II which synthesizes mRNA precursors and many functional non-coding RNAs. Forms the polymerase active center together with the second largest subunit. Pol II is the central component of the basal RNA polymerase II transcription machinery. It is composed of mobile elements that move relative to each other. RPB1 is part of the core element with the central large cleft, the clamp element that moves to open and close the cleft and the jaws that are thought to grab the incoming DNA template. At the start of transcription, a single-stranded DNA template strand of the promoter is positioned within the central active site cleft of Pol II. A bridging helix emanates from RPB1 and crosses the cleft near the catalytic site and is thought to promote translocation of Pol II by acting as a ratchet that moves the RNA-DNA hybrid through the active site by switching from straight to bent conformations at each step of nucleotide addition. During transcription elongation, Pol II moves on the template as the transcript elongates. Elongation is influenced by the phosphorylation status of the C-terminal domain (CTD) of Pol II largest subunit (RPB1), which serves as a platform for assembly of factors that regulate transcription initiation, elongation, termination and mRNA processing. Regulation of gene expression levels depends on the balance between methylation and acetylation levels of tha CTD-lysines (By similarity). Initiation or early elongation steps of transcription of growth-factors-induced immediate early genes are regulated by the acetylation status of the CTD (PubMed:24207025). Methylation and dimethylation have a repressive effect on target genes expression
Whole cell extracts (40 μg) from HeLa cells transfected with Pol II siRNA (lane 2) and from an untransfected control (lane 1) were analysed by WB using the Pol II Monoclonal Antibody (bsm-53004M) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk, followed by secondary antibody incubation.
Nuclear extracts (25 μg) from HeLa cells were analysed by Western blot using the Pol II Monoclonal Antibody (bsm-53004M) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk, followed by secondary antibody incubation.
HeLa cells were stained with the Pol II Monoclonal Antibody (bsm-53004M) and DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were labelled with antibody (left) at 1:500 dilution in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two staining is shown on the right.
To test the specificity an ELISA was performed using Pol II Monoclonal Antibody(bsm-53004M). The wells were coated with peptides containing the unmodified C-terminal repeat sequence as well as different phosphorylated peptides. Figure shows that the antibody recognizes the unphosphorylated Pol II as well as most phosphorylated forms.