Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095)

  Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody (ab5095)

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 1/500 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 ab5095, blue by DAPI. Please note that there may be variability of staining patterns depending on the cell type.

Michael Mancini, Baylor College of Medicine

  ChIP - RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095)

ab5095 at 4ug/ml in ChIP of RAW macrophages. Nuclear cell lysate of mouse RAW macrophages (expressing c-fms) were formaldehyde cross linked and ChIP tested with ab5095. The nuclear preparation was frozen before sonication with a probe sonicator. All buffers used contained protease inhibitors. 3T3 fibroblasts (not expressing c-fms) were used as the negative control.

This image is courtesy of an anonymous Abreview

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  ChIP - RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095)

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 25 µg of chromatin, 2µg of  ab5095 (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, a region in the GAPDH gene (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.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095)

Image courtesy of Human Protein Atlas

ab5095 staining in human brain, showing staining of the Purkinje cells (in brown). Paraffin embedded brain tissue was incubated with ab5095 (1:900 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

ab5095 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

  Western blot - RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095)

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : S.cerevisiae (Y190) Whole Cell Lysate
Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell 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) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (ab12793) at 1 µg/ml
Lane 6 : S.cerevisiae (Y190) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (ab12793) at 1 µg/ml
Lane 7 : 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 8 : S.cerevisiae (Y190) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488) at 1 µg/ml

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 217 kDa
Observed band size : 240 kDa (why is the actual band size different from the predicted?)

  Immunocytochemistry/ Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095)

ICC/IF image of ab5095 stained MCF7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab5095, 1µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.