All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [EPR19015] (ab193467) at 1/5000 dilution

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

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: 5 seconds

Blocking/Dilution buffer: 2% BSA/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized PC-12 (Rat adrenal gland pheochromocytoma cell line) cells labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab193467 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on PC-12 cell line. The expression decreased after treatment with Lambda Phosphatase at 31°C for 5h.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab193467 at 1/250 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab193467 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on epithelial 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.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab193467 at 1/30 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

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

Labeled using ab193467 at 1/1000 dilution (0.1 µg/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.

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 S5) with ab193467 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cell line. The expression decreased after treatment with Lambda Phosphatase 31°C for 5h.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab193467 at 1/250 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution 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 S5) antibody [EPR19015] (ab193467) at 1/1000 dilution

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

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: 5 seconds

Blocking/Dilution buffer: 2% BSA/TBST.

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [EPR19015] (ab193467) at 1/1000 dilution

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

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: 1 second

Blocking/Dilution buffer: 2%BSA/TBST.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab193467 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on lymphocytes of 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.

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 S5) with ab193467 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on RAW 264.7 cell line. The expression decreased after treatment with Lambda Phosphatase 31°C for 5h.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab193467 at 1/250 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

RNA polymerase II CTD repeat YSPTSPS (phospho S5) was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab193467 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab193467 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: ab193467 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab193467 in HeLa whole cell lysate.

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

Dot blot analysis of RNA polymerase II CTD repeat YSPTSPS (phospho S5) phospho peptide (Lane 1) and RNA polymerase II CTD repeat YSPTSPS non-phospho peptide (Lane 2) labeled using ab193467 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1/1000 dilution.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.