ChIP - RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)

Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab5408 (blue), and 20 µl of protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

Western blot - RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide (ab17564) at 1 µg/ml
Lane 3 : 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 4 : HeLa (Human epithelial carcinoma 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/ml per lane.

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 217 kDa
Observed band size : 260 kDa (why is the actual band size different from the predicted?)


Exposure time : 30 seconds

Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)

HeLa or MCF7 cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/1000 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-mouse Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in TBS-T. Coverslips were then counterstained with DAPI in TBS-T for 1-2 minutes, TBS-T was then added and the coverslips mounted. Red indicates staining by ab5408, blue by DAPI.

Michael Mancini, Baylor College of Medicine

Western blot - RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)

Predicted band size : 217 kDa

Lane 1: buffer,

Lane 2: extract from human 293T cells as positive control,

Lanes 3 and 4: egg extracts made from wild type N2 worms and worms grown on RNAi food against dicer,

Lanes 5-8: intact worms in mentioned larval stage and eggs boiled directly in SDS-PAGE buffer and loaded

This image was kindly supplied as part of the review submitted by Shveta Bagga.

ELISA - RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)

ELISA using ab5408 at varying antibody concentrations. Curve_SPL4 indicates binding to RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488). Binding to the following peptides was much weaker: Curve_SPL5 RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (ab12793), Curve_SPL6 RNA polymerase II CTD repeat YSPTSPS peptide (ab12795).

Flow Cytometry-Anti-RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade(ab5408)

Overlay histogram showing HeLa cells stained with ab5408 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab5408, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.