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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).|
|IHC-P||1/500. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|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.|
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.
Image courtesy of an anonymous AbReview
Immunohistochemical analysis of paraffin-embedded human urinary bladder tissue labelingRNA polymerase II CTD repeat YSPTSPS with ab26721 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab) at 1/1000 dilution (non-abcam). Nuclear staining on human urinary bladder is observed.
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.
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.