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Our Abpromise guarantee covers the use of ab26721 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 220 kDa (predicted molecular weight: 220 kDa).|
|IP||Use a concentration of 5 µg/ml.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|ChIP||Use 5-10 µg for 25 µg of chromatin.|
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 70oC; 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.
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.
ICC/IF image of ab26721 stained human HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab26721, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HEK 293, HepG2 and MCF7 cells.
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.
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