Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 4 µg of Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855-87] - ChIP Grade (ab238146). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

Additional screenshots of mapped reads can be downloaded here.

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab238146 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat testis without alkaline phosphatase treatment (panel A). No signal was detected when treated with alkaline phosphatase (panel B). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab238146 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 30 µg of chromatin and 4 µg of Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855-87] - ChIP Grade (ab238146). ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 30 million reads. ChIP-Seq validation performed by Active Motif, Carlsbad, CA.

Additional screenshots of mapped reads can be downloaded here.

immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab238146 at 1/500 dilution, followed by a Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in RAW 264.7 cell line, the signal decreased after phosphatase treatment at 37℃ for 2h. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Chromatin was prepared from HeLa (human epithelial cell line from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10min.

The ChIP was performed with 25 µg of chromatin, 5 µg of ab238146 (red), and 20 µl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (sybr green approach).

Primers and probes are located in the first kb of the transcribed region.

RNA polymerase II CTD repeat YSPTSPS (phospho S2) was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab238146 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab238146 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 μg (Input).
Lane 2: ab237146 IP in HeLa whole cell lysate.
Lane 3: : Rabbit monoclonal IgG (ab172730) instead of ab238146 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 7 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab238146 at 1/600 (red) compared with a Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855-87] - ChIP Grade (ab238146) at 1/5000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa whole cell lysate (phosphatase-treated membrane)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 217 kDa
Observed band size: 270 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Dot blot analysis of RNA polymerase II CTD repeat YSPTSPS (phospho S2) labeled with ab238146 at 1/1000 dilution.

Lane 1: RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide.
Lane 2: RNA polymerase II CTD repeat YSPTSPS non-phospho peptide.
Lane 3: RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide.

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab238146 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse testis without alkaline phosphatase treatment (panel A). No signal was detected when treated with alkaline phosphatase (panel B). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab238146 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab238146 at 1/2000 dilution, followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human testis without alkaline phosphatase treatment (panel A). No signal was detected when treated with alkaline phosphatase (panel B). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab238146 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab238146 at 1/500 dilution, followed by a Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in HeLa cell line, the signal decreased after phosphatase treatment at 37℃ for 2h. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855-87] - ChIP Grade (ab238146) at 1/2000 dilution

Lane 1 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : RAW 264.7 whole cell lysate (phosphatase-treated membrane)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 217 kDa
Observed band size: 270 kDa why is the actual band size different from the predicted?


Exposure time: 37 seconds

Blocking and dilution buffer: 1% BSA/TBST.

The molecular weight  observed is consistent with what has been described in the literature (PMID: 25849141).

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855-87] - ChIP Grade (ab238146) at 1/2000 dilution

Lane 1 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 2 : PC-12 whole cell lysate (phosphatase-treated membrane)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 217 kDa
Observed band size: 270 kDa why is the actual band size different from the predicted?


Exposure time: 37 seconds

Blocking and dilution buffer: 1% BSA/TBST.