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 ab252852 (red), or 5 µg of Rat IgG2a ab18450 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab252852 at 1/500 (1.29 μg/mL) dilution , followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 2 dilution (Green). Confocal image showing nuclear staining in HeLa cell line, the signal decreased after phosphatase treatment at 37°C for 2h. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/500 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at 1/500 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.

RNA polymerase II CTD repeat YSPTSPS (phospho S5) labeled with ab252852 at 1/1000 (0.645 μg/mL) dilution.

Goat Anti-Rat IgG (H+L), HRP) (ab205720) at 1/5000 dilution was used as secondary antibody.

Lane 1: RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide

Lane 2: RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide

Lane 3: RNA polymerase II CTD repeat YSPTSPS (phospho T4) peptide

Lane 4: RNA polymerase II CTD repeat YSPTSPS (phospho Y1) peptide

Lane 5: RNA polymerase II CTD repeat YSPTSPS (phospho S7) peptide

Lane 6: RNA polymerase II CTD repeat YSPTSPS non-phopho peptide

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [3E8] - ChIP Grade (ab252852) at 0.645 µg/ml

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: 3 seconds.

ab252852 was used at 0 - 1000 ng/mL.

Antigens were used at 1000 ng/mL.

An Alkaline Phosphatase-conjugated Anti-Rat IgG (H+L) wasused as secondary antibody at 1/1000 dilution.

 

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [3E8] - ChIP Grade (ab252852) at 0.645 µg/ml

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
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: 3 seconds.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW264.7 cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab252852 at 1/500 (1.29 μg/mL) 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 RAW264.7 cell line, the signal decreased after phosphatase treatment at 37°C for 2h. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/500 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) secondary antibody at 1/500 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 S5) antibody [3E8] - ChIP Grade (ab252852) at 0.645 µg/ml

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: 37 seconds.