Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)

ab26721 stained in HeLa cells. Cells were fixed with 4% paraformaldehyde (10 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab26721 at 1µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

IHC image of RNA polymerase II CTD repeat YSPTSPS staining in a section of formalin-fixed paraffin-embedded normal human Hodgkin's lymphoma* performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab26721, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.*

Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25µg of chromatin, 5µg of  ab26721 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the inactive AFM and F8 promoters, the GAPDH promoter (active) and over the g-Actin gene (active). Schematic diagram of the g-Actin gene is shown on the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene.
   

RNA polymerase II CTD repeat YSPTSPS was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to RNA polymerase II CTD repeat YSPTSPS and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab26721.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to mouse anti-Rabbit HRP (IgG light chain) (ab99697).
Band: 220kDa; RNA polymerase II CTD repeat YSPTSPS.

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ab26721 staining RNA polymerase II CTD in the human HeLa cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Tween-20 and blocked with 1% BSA for 10 minutes at 25°C. Samples were incubated with primary antibody (1/400 in TBS) for 12 hours at 4°C. A FITC conjugated anti-rabbit goat IgG polyclonal (1/500) was used as the secondary antibody.

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