Recombinant
RabMAb

Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855] (ab193468)

Overview

  • Product name
    Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [EPR18855]
    See all RNA polymerase II CTD repeat YSPTSPS primary antibodies
  • Description
    Rabbit monoclonal [EPR18855] to RNA polymerase II CTD repeat YSPTSPS (phospho S2)
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IF, IP, Flow Cyt, Dot blotmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Saccharomyces cerevisiae
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Saccharomyces cerevisiae RNA polymerase II CTD repeat YSPTSPS aa 1600-1700 (phospho S2) (Cysteine residue). The exact sequence is proprietary.
    Database link: P04050

  • Positive control
    • WB: MCF7, HeLa, RAW 264.7 and PC-12 whole cell lysates. IHC-P: Mouse kidney, spleen and testis tissues. ICC/IF: HeLa, MCF7 and PC-12 cells. IP: HeLa whole cell lysate.
  • General notes

    This product is a recombinant rabbit monoclonal antibody.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

Applications

Our Abpromise guarantee covers the use of ab193468 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/5000. Detects a band of approximately 270 kDa (predicted molecular weight: 192 kDa).
IHC-P 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Recommended for mouse only.

ICC/IF 1/100.
IP 1/50.
Flow Cyt Use at an assay dependent concentration.
Dot blot 1/1000.

Target

  • Function
    DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. Largest and catalytic component of RNA polymerase II which synthesizes mRNA precursors and many functional non-coding RNAs. Forms the polymerase active center together with the second largest subunit. Pol II is the central component of the basal RNA polymerase II transcription machinery. It is composed of mobile elements that move relative to each other. RPB1 is part of the core element with the central large cleft, the clamp element that moves to open and close the cleft and the jaws that are thought to grab the incoming DNA template. At the start of transcription, a single-stranded DNA template strand of the promoter is positioned within the central active site cleft of Pol II. A bridging helix emanates from RPB1 and crosses the cleft near the catalytic site and is thought to promote translocation of Pol II by acting as a ratchet that moves the RNA-DNA hybrid through the active site by switching from straight to bent conformations at each step of nucleotide addition. During transcription elongation, Pol II moves on the template as the transcript elongates. Elongation is influenced by the phosphorylation status of the C-terminal domain (CTD) of Pol II largest subunit (RPB1), which serves as a platform for assembly of factors that regulate transcription initiation, elongation, termination and mRNA processing. Acts as an RNA-dependent RNA polymerase when associated with small delta antigen of Hepatitis delta virus, acting both as a replicate and transcriptase for the viral RNA circular genome.
  • Sequence similarities
    Belongs to the RNA polymerase beta' chain family.
  • Domain
    The C-terminal domain (CTD) serves as a platform for assembly of factors that regulate transcription initiation, elongation, termination and mRNA processing.
  • Post-translational
    modifications
    The tandem heptapeptide repeats in the C-terminal domain (CTD) can be highly phosphorylated. The phosphorylation activates Pol II. Phosphorylation occurs mainly at residues 'Ser-2' and 'Ser-5' of the heptapeptide repeat and is mediated, at least, by CDK7 and CDK9. CDK7 phosphorylation of POLR2A associated with DNA promotes transcription initiation by triggering dissociation from DNA. Phosphorylation also takes place at 'Ser-7' of the heptapeptide repeat, which is required for efficient transcription of snRNA genes and processing of the transcripts. The phosphorylation state is believed to result from the balanced action of site-specific CTD kinases and phosphatases, and a 'CTD code' that specifies the position of Pol II within the transcription cycle has been proposed. Dephosphorylated by the protein phosphatase CTDSP1.
    Among tandem heptapeptide repeats of the C-terminal domain (CTD) some do not match the Y-S-P-T-S-P-S consensus, the seventh serine residue 'Ser-7' being replaced by a lysine. 'Lys-7' in these non-consensus heptapeptide repeats can be alternatively acetylated, methylated and dimethylated. EP300 is one of the enzyme able to acetylate 'Lys-7'. Acetylation at 'Lys-7' of non-consensus heptapeptide repeats is associated with 'Ser-2' phosphorylation and active transcription. It may regulate initiation or early elongation steps of transcription specially for inducible genes.
    Methylated at Arg-1810 prior to transcription initiation when the CTD is hypophosphorylated, phosphorylation at Ser-1805 and Ser-1808 preventing this methylation. Symmetrically or asymmetrically dimethylated at Arg-1810 by PRMT5 and CARM1 respectively. Symmetric or asymmetric dimethylation modulates interactions with CTD-binding proteins like SMN1/SMN2 and TDRD3. SMN1/SMN2 interacts preferentially with the symmetrically dimethylated form while TDRD3 interacts with the asymmetric form. Through the recruitment of SMN1/SMN2, symmetric dimethylation is required for resolving RNA-DNA hybrids created by RNA polymerase II, that form R-loop in transcription terminal regions, an important step in proper transcription termination. CTD dimethylation may also facilitate the expression of select RNAs. Among tandem heptapeptide repeats of the C-terminal domain (CTD) some do not match the Y-S-P-T-S-P-S consensus, the seventh serine residue 'Ser-7' being replaced by a lysine. 'Lys-7' in these non-consensus heptapeptide repeats can be alternatively acetylated, methylated and dimethylated. Methylation occurs in the earliest transcription stages and precedes or is concomitant to 'Ser-5' and 'Ser-7' phosphorylation.
    Ubiquitinated by WWP2 leading to proteasomal degradation (By similarity). Following UV treatment, the elongating form of RNA polymerase II (RNA pol IIo) is ubiquitinated UV damage sites without leading to degradation: ubiquitination is facilitated by KIAA1530/UVSSA and promotes RNA pol IIo backtracking to allow access to the nucleotide excision repair machinery.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • DNA directed RNA polymerase II A antibody
    • DNA-directed RNA polymerase II largest subunit RNA polymerase II 220 kd subunit antibody
    • DNA-directed RNA polymerase II subunit A antibody
    • DNA-directed RNA polymerase II subunit RPB1 antibody
    • DNA-directed RNA polymerase III largest subunit antibody
    • hRPB220 antibody
    • hsRPB1 antibody
    • POLR2 antibody
    • Polr2a antibody
    • POLRA antibody
    • Polymerase (RNA) II (DNA directed) polypeptide A 220kDa antibody
    • Polymerase (RNA) II (DNA directed) polypeptide A antibody
    • RNA polymerase II subunit B1 antibody
    • RNA-directed RNA polymerase II subunit RPB1 antibody
    • RPB1 antibody
    • RPB1_HUMAN antibody
    • RPBh1 antibody
    • RpIILS antibody
    • RPO2 antibody
    • RPOL2 antibody
    see all

Images

  • 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
    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.

  • 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.

  • 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.

  • 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.

  • 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
    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.

  • 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.

  • 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
    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
    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.

  • 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.

     

  • 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.

  • 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.

  • 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.

  • 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 secondary antibody (HRP) (ab131366), was used as secondary antibody 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.

References

This product has been referenced in:
  • Zhang X  et al. Peptidylarginine deiminase 1-catalyzed histone citrullination is essential for early embryo development. Sci Rep 6:38727 (2016). ICC/IF ; Rat . Read more (PubMed: 27929094) »

See 1 Publication for this product

Customer reviews and Q&As

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Zebrafish Cell (Dissociated gastrula embryo cells)
Permeabilization
Yes - 0.5% Triton x-100, 10 minutes
Specification
Dissociated gastrula embryo cells
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 4% · Temperature: 25°C
Fixative
Formaldehyde
Username

Dr. Lennart Hilbert

Verified customer

Submitted Dec 20 2016

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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