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

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.
   

Immunocytochemistry/ Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)

ab26721 (1/200) staining RNA polymerase II in assyncchonous HeLa cells (green). cells were fixed in paraformaldehyde, permeabilised in 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

Image courtesy of an abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

Immunocytochemistry/ Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)

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.

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

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide (ab17564) at 1 µg/ml
Lane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide (ab17564) at 1 µg/ml
Lane 5 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488) at 1 µg/ml
Lane 6 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488) at 1 µg/ml
Lane 7 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (ab12793) at 1 µg/ml
Lane 8 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (ab12793) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 220 kDa
Observed band size : 220 kDa


Exposure time : 1 minute