Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/500 (1.0 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing nuclear staining in HeLa cell line (shown in green), the signal decreased after lambda protein phosphatase treatment at 37°C for 2h. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/5000 (0.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human cerebrum without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab317456 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/5000 (0.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab317456 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) treated with alkaline phosphatase (Right) / Untreated HeLa (Left) cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/5000 dilution (0.1ug) (Red) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

RNA polymerase II CTD repeat YSPTSPS (phospho S5) was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab317456 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317456 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : ab317456 IP in HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317456 in HeLa whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (ab317456) at 1/30 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Lane 2:

ab317456 IP in HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 6s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/5000 (0.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat cerebrum without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab317456 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized PC-12 (rat adrenal gland pheochromocytoma cell) cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/500 (1.0 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing nuclear staining in PC-12 cell line (shown in green), the signal decreased after lambda protein phosphatase treatment at 37°C for 2h. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/5000 (0.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse colon without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab317456 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/5000 (0.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat colon without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab317456 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/5000 (0.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab317456 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with alkaline phosphatase (Right) / Untreated RAW 264.7 (Left) cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/5000 dilution (0.1ug) (Red) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized PC-12 (rat adrenal gland pheochromocytoma cell) treated with alkaline phosphatase (Right) / Untreated PC-12 (Left) cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/5000 dilution (0.1ug) (Red) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab317456 at 1/500 (1.0 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing nuclear staining in RAW 264.7 cell line (shown in green), the signal decreased after lambda protein phosphatase treatment at 37°C for 2h. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

RNA polymerase II CTD repeat YSPTSPS (phospho S5) was immunoprecipitated from 0.35 mg PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate with ab317456 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317456 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate
Lane 2 : ab317456 IP in PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317456 in PC-12 whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (ab317456) at 1/30 dilution

Lane 1:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate

Lane 2:

ab317456 IP in PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 6s

• IP

Supplier Data

Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

RNA polymerase II CTD repeat YSPTSPS (phospho S5) was immunoprecipitated from 0.35 mg RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate with ab317456 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317456 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 2 : ab317456 IP in RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317456 in RAW 264.7 whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (ab317456) at 1/30 dilution

Lane 1:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

Lane 2:

ab317456 IP in RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

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

In Western blot, Anti-RNA polymerase II CTD repeat YSPTSPS antibody [EPR24494-59] staining at ChIP Grade dilution.

All lanes:

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (ab317456) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate (untreated membrane) at 20 µg

Lane 3:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate (untreated membrane) at 20 µg

Lane 4:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate (untreated membrane) at 20 µg

Lane 5:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 6:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 7:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 8:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 270 kDa,124 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

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

For Lane 3, this blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

Exposure time :

Lane 1-2 : 114 seconds

Lane 3 : 48 seconds

All lanes:

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (ab317456) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 270 kDa

true

• Dot

Supplier Data

Dot Blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (AB317456)

Dot blot analysis of RNA polymerase II CTD repeat YSPTSPS (phospho S5) using ab317456 at 1 : 1000 dilution (0.5 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Exposure time : 180 seconds.

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

All lanes:

Dot Blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [RM1152] (ab317456) at 1/1000 dilution

Lane 1:

RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide

Lane 2:

RNA polymerase II CTD repeat YSPTSPS non-phospho peptide

Lane 3:

RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide

Lane 4:

RNA polymerase II CTD repeat YSPTSPS (phospho Y1) peptide

Secondary

All lanes:

Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

Exposure time: 180s