Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)

  Western blot - RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lane 2 : S.cerevisiae (Y190) Whole Cell Lysate
Lane 3 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate with RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml
Lane 4 : S.cerevisiae (Y190) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml
Lane 5 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488) at 1 µg/ml
Lane 6 : S.cerevisiae (Y190) 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) Nuclear Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (ab12793) at 1 µg/ml
Lane 8 : S.cerevisiae (Y190) 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) at 1/3000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 217 kDa


Exposure time : 30 seconds

  ChIP - RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)

Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 mins. The  ChIP was performed with 25 µg of chromatin, 2 µg of  ab5131 (blue), and 20 µl 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 y-Actin gene (active). Schematic diagram of the y-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.

  Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)

Lanes 1, 3 & 4 : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131) at 1/500 dilution
Lane 2 : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131) at 1/2000 dilution

Lane 1 : HeLa nuclear extract
Lane 2 : HeLa nuclear extract
Lane 3 : HeLa nuclear extract with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488) at 1 µg/ml
Lane 4 : HeLa nuclear extract with RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 217 kDa
Observed band size : 250 kDa (why is the actual band size different from the predicted?)

  Immunocytochemistry/ Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)

ICC/IF image of ab5131 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab5131, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and 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).

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)

(Image courtesy of Human Protein Atlas) ab5131 staining RNA Polymerase in Human urinary bladder tissue. Paraffin embedded human skin tissue was incubated with ab5131 for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. ab5131 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

  Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (ab5131)

HeLa 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 1/160 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-rabbit 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 ab5131, blue by DAPI.

Michael Mancini, Baylor College of Medicine