ChIP assays were performed using the breast carcinoma cell line MCF7 (treated with estradiol (MCF7 + E2) or not treated (MCF7)), the anti–Pol II antibody (Cat. no. 49-1033), and optimized PCR primer sets for myoglobin exon 2, c-fos promoter, β-actin promoter for qPCR. Each ChIP assay used sheared chromatin from 1 million cells and 2.8 µl of anti-Pol II ascites. Pol II binds to active promoters of protein-coding genes. Primer pairs for the following promoter regions were used: c-fos and β-actin. Myoglobin exon 2, which is not a promoter region, was used as negative PCR control. Recoveries (% of input) are shown here above. The percent recovery represents the relative amount of immunoprecipitated DNA compared to input DNA. The recoveries are high using the positive primer pairs c-fos promoter (bars 2 and 5) and β-actin promoter (bars 3 and 6), while there are no recoveries using the negative primer pair myoglobin exon 2 (bars 1 and 4). Moreover, recoveries are higher in cells treated with estradiol (MCF7 + E2) compared to recoveries in untreated MCF7 cells as expected.
|Tested species reactivity||Human|
|Host / Isotype||Mouse / IgG2a|
|Immunogen||Raised against the largest subunit of RNA polymerase II of wheat germ and interacts with the highly conserved C-terminal domain of this protein containing the repeat YSPTSPS.|
|Contains||<0.1% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||2-3 ul|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
This gene encodes the largest subunit of RNA polymerase II, the polymerase responsible for synthesizing messenger RNA in eukaryotes. The product of this gene contains a carboxy terminal domain composed of heptapeptide repeats that are essential for polymerase activity. These repeats contain serine and threonine residues that are phosphorylated in actively transcribing RNA polymerase. In addition, this subunit, in combination with several other polymerase subunits, forms the DNA binding domain of the polymerase, a groove in which the DNA template is transcribed into RNA.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.