Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)

Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab26721 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 Use a concentration of 1 µg/ml. Detects a band of approximately 220 kDa (predicted molecular weight: 220 kDa).Can be blocked with Human RNA polymerase II CTD repeat YSPTSPS peptide (ab17564).
IP Use a concentration of 5 µg/ml.
ICC/IF Use a concentration of 1 µg/ml.
ChIP Use 5-10 µg for 25 µg of chromatin.

Target

  • FunctionDNA-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 a 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 similaritiesBelongs to the RNA polymerase beta' chain family.
  • Post-translational
    modifications
    The tandem 7 residues 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 heptapepdtide 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.
    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.
    Methylated at Arg-1810 by CARM1. Methylation occurs only when the CTD is hypophosphorylated, and phosphorylation at Ser-1805 and Ser-1808 prevent methylation (in vitro). It is assumed that methylation occurs prior to phosphorylation and transcription initiation. CTD methylation may facilitate the expression of select RNAs.
  • Cellular localizationNucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • DNA directed RNA polymerase II A antibody
    • DNA-directed RNA polymerase II largest subunit 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 pol II CTD 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

Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade images

  • Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25µg of chromatin, 5µg of  ab26721 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the inactive AFM and F8 promoters, the GAPDH promoter (active) and over the g-Actin gene (active). Schematic diagram of the g-Actin gene is shown on the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene.
       

  • ab26721 (1/200) staining RNA polymerase II in assyncchonous HeLa cells (green). cells were fixed in paraformaldehyde, permeabilised in 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

    See Abreview

  • ICC/IF image of ab26721 stained human HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab26721, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HEK 293, HepG2 and MCF7 cells.

  • All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
    Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Human RNA polymerase II CTD repeat YSPTSPS peptide (ab17564) at 1 µg/ml
    Lane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with Human RNA polymerase II CTD repeat YSPTSPS peptide (ab17564) at 1 µg/ml
    Lane 5 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488) at 1 µg/ml
    Lane 6 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488) at 1 µg/ml
    Lane 7 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (ab12793) at 1 µg/ml
    Lane 8 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (ab12793) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 220 kDa
    Observed band size : 220 kDa


    Exposure time : 1 minute
  • RNA polymerase II CTD repeat YSPTSPS was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to RNA polymerase II CTD repeat YSPTSPS and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab26721.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 220kDa; RNA polymerase II CTD repeat YSPTSPS.

References for Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)

This product has been referenced in:
  • Fonseca GJ  et al. Adenovirus E1A recruits the human Paf1 complex to enhance transcriptional elongation. J Virol 88:5630-7 (2014). Human . Read more (PubMed: 24600005) »
  • Yin T  et al. A novel CDK9 inhibitor shows potent antitumor efficacy in preclinical hematologic tumor models. Mol Cancer Ther 13:1442-56 (2014). Read more (PubMed: 24688048) »

See all 6 Publications for this product

Product Wall

Application ChIP
Detection step Real-time PCR
Sample Human Cell lysate - nuclear (Prostate)
Specification Prostate
Negative control An unspecified region 10000kb downstream of the putative binding spot.
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1.0% Formaldehyde
Positive control Pol II served as my positive control and tested a TSS where another paper has confirmed via ChIP-chip that Pol II binds there.
Username

Mr. Vineet Dhiman

Verified customer

Submitted Oct 17 2013

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (10%)
Sample Mouse Cell lysate - whole cell (MEL)
Specification MEL
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Oct 31 2013

Ab26721 is indeed specific to non-phosphorylated RNA Pol II. It's immunogen is proprietary but I can share this information with you to help you with your research as long as you do not publish it. Ab26721 immuogen is a synthetic peptide of Human RNA p...

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Merci de nous avoir contactés.

Le numéro de commande pour ab26721 et ab1791 est xxxxxxxx. Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

Le numéro de com...

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Merci de nous avoir contactés.

Nous sommes désolés d'apprendre que les produits que vous avez reçus ne fonctionnent pas comme attendu. Je me permets de vous transférer le numéro de commande des rempl...

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Merci de nous avoir contactés.

Nous sommes désolés d'apprendre que les produits que vous avez reçus ne fonctionnent pas comme attendu. Je suis d’accord pour vous envoyer ab26721, ab106293 et ab1791 en rempla...

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Merci de nous avoir contactés.

J’ai eu ma réponse de nos laboratoires et ce cas et assez compliqué. Premièrement je tiens à vous remercier d’avoir patienté et j’apprécie &eacu...

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Thank you for contacting us and your interest in our products.

I have had a look into your question of whether the Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721) is likely to be able to detect both the phosphorylate...

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Thank you for contacting Abcam earlier today.
As we discussed, I am sending you new aliquots of both ab24552 and ab26721 and your new order number is ***********.
Below are a few possible alternatives if the replacements are not successful: <...

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Thank you for contacting us.

We have tested some of our RNA polymerase II antibodies against both inactive genes and the 3' end of active genes. Below is a link to the antibodies.

http://www.abcam.com/RNA-polymerase-II-CTD-repeat-...

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