RNA polymerase II CTD repeat YSPTSPS (phospho S2) was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab193468 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab193468 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HeLa whole cell lysate 10µg (Input).

Lane 2: ab193468 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab193468 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.

Chromatin was prepared from HeLa cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 μg of chromatin, 5 μg of ab193468 (red), or 5 μg of rabbit normal IgG ab172730 (gray) and 20 μL of Protein A/G sepharose beads. 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.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab193468 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mouse spleen is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab193468 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on epithelium cells and glomerulus cells of mouse kidney is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab193468 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mouse testis is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855] (ab193468) at 1/5000 dilution

Lane 1 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : MCF7 whole cell lysate treated with Lambda Phosphatase

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


Exposure time: 3 seconds

Blocking/Dilution buffer: 1% BSA/TBST.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855] (ab193468) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa whole cell lysate treated with Lambda Phosphatase

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


Exposure time: 3 seconds

Blocking/Dilution buffer: 1% BSA/TBST.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855] (ab193468) at 1/5000 dilution

Lane 1 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 2 : RAW 264.7 whole cell lysate treated with Lambda Phosphatase

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


Exposure time: 3 seconds

Blocking/Dilution buffer: 1% BSA/TBST.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855] (ab193468) at 1/5000 dilution

Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 2 : PC-12 whole cell lysate treated with Lambda Phosphatase

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


Exposure time: 3 seconds

Blocking/Dilution buffer: 1% BSA/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) cells labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab193468 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on MCF7 cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab193468 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab193468 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab193468 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized PC-12 (Rat adrenal gland pheochromocytoma) cells labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab193468 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on PC-12 cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:-

-ve control 1: ab193468 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.

-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling RNA polymerase II CTD repeat YSPTSPS antibody (phospho S2) (red) with purified ab193468 at a dilution of 1/70. Goat anti rabbit IgG (Alexa Fluor® 488) was used as the secondary antibody at 1/2000. Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Isotype control antibody was Rabbit monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody.

Dot blot analysis of RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (Lane 1) and non-phospho peptide (Lane 2) labeled using ab193468 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody (ab97051) at 1/100000 dilution.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

Lane 1: RNA polymerase II CTD repeat YSPTSPS (phospho S2) phospho peptide
Lane 2: RNA polymerase II CTD repeat YSPTSPS non-phospho peptide (peptide of ab193468)
Lane 3: RNA polymerase II CTD repeat YSPTSPS (phospho S5) phospho peptide
Lane 4: RNA polymerase II CTD repeat YSPTSPS non-phospho peptide (peptide of ab193467)

Labeled using ab193468 at 1/1000 dilution (0.8 ug/ml), followed by Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1/100000 dilution.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.