Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S7) with ab252853 at 1/100 dilution, followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cells. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) at 1/1000  dilution.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S7) antibody [4E12] (ab252853) at 1/1000 dilution

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

Lysates/proteins at 10 µg per lane.

Secondary
Lane 1 : Goat Anti-Rat IgG (H+L), HRP) (ab205720) at 1/5000 dilution
Lane 2 : Goat Anti-Rat IgG H&L (HRP) (ab205720) at 1/5000 dilution

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

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile/ molecular weight  observed is consistent with what has been described in the literature (PMID: 22745433 and 23071310)

Exposure time: 10 seconds

RNA polymerase II CTD repeat YSPTSPS (phospho S7) was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate with ab252853 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab252853 at 1/1000 dilution. Goat Anti-Rat IgG (H+L), HRP) (ab205720) was used at 1/10000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate 10 ug

Lane 2: ab252853 IP in HeLa whole cell lysate

Lane 3: Rat monoclonal IgG1 (ab18407) instead of ab252853 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 ab252853 (red), or 5 µg of Rat IgG1 ab18407 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

ELISA analysis using ab252853 at a range of 1000-0 ng/ml followed by a Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rat IgG (H+L) at 1/1000 dilution. Antigen concentration: 100 ng/ml

Antigens: phospho S2, phospho S5, phospho T4, phospho Y1, phospho S7, non-phospho

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S7) with ab252853 at 1/100 dilution, followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in RAW 264.7 cells. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S7) antibody [4E12] (ab252853) at 1/1000 dilution

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate (Untreated membrane)
Lane 2 : RAW264.7 whole cell lysate (Phosphatase treated membrane)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rat IgG (H+L), HRP) (ab205720) at 1/5000 dilution

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

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile/ molecular weight  observed is consistent with what has been described in the literature (PMID: 22745433 and 23071310) 

Exposure time: 20 seconds

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S7) antibody [4E12] (ab252853) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate (Untreated membrane)
Lane 2 : HeLa whole cell lysate (Phosphatase treated membrane)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rat IgG (H+L), HRP) (ab205720) at 1/5000 dilution

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

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile/ molecular weight  observed is consistent with what has been described in the literature (PMID: 22745433 and 23071310)

Exposure time: 20 seconds