Anti-RNA polymerase II CTD repeat YSPTSPS antibody [8WG16] - ChIP Grade (ab817)

Overview

  • Product nameAnti-RNA polymerase II CTD repeat YSPTSPS antibody [8WG16] - ChIP Grade
    See all RNA polymerase II CTD repeat YSPTSPS primary antibodies
  • Description
    Mouse monoclonal [8WG16] to RNA polymerase II CTD repeat YSPTSPS - ChIP Grade
  • SpecificityThis antibody reacts with the highly conserved heptapeptide repeat of the largest subunit of eukaryotic RNA polymerase II.
  • Tested applicationsCHIPseq, ICC, Purification, WB, ICC/IF, IHC - Wholemount, ChIP, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Mouse, Cow, Human, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Quail, Neurospora crassa , Rice
    Predicted to work with: a wide range of other species
  • Immunogen

    Purified wheat germ RNA polymerase II.

  • General notesThis antibody may be used to detect unproteolyzed RNA polymerase II or to inhibit specific transcription from class II promoters. This is a polyol-responsive antibody and can be used in gentle purifications of RNA polymerase II from from wheat germ, calf thymus, and yeast and is likely to purify RNA polymerase II from most eukaryotic organisms. It inhibits promoter-directed transcription, but it does not inhibit elongation in the nonspecific transcription assay.

Properties

Applications

Our Abpromise guarantee covers the use of ab817 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
CHIPseq Use at an assay dependent concentration. PubMed: 19251593Use 2ug per 0.3ml of sonicated chromatin.
ICC Use at an assay dependent concentration.
Purification Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Detects a band of approximately 217 kDa (predicted molecular weight: 217 kDa).
ICC/IF Use at an assay dependent concentration.
IHC - Wholemount Use at an assay dependent concentration.

See Abreview.

ChIP Use at an assay dependent concentration. See Abreview; the user recommends using anti-mouse IgG coated Dynabeads instead of Protein A to recover the precipitate).
Flow Cyt Use 0.5µg for 106 cells.
IP Use at an assay dependent concentration.

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
    see all
  • 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 [8WG16] - ChIP Grade images

  • Various regions across the Actin2/7 loci were tested for the presence of RNA polymerase II CTD repeat YSPTSPS. A nuclear lysate from Arabidopsis thaliana seedlings was crosslinked using 1% formaldehyde for 30 seconds.  The ChIP was performed with 0.1 µg of ab817 per µg of chromatin; incubated together for 16 hours at 4°C.  The immunoprecipitated DNA was quantified by Real-Time PCR.  The bottom panel indicates the positive (ab817) and negative controls (no antibody) at region B3.

    See Abreview

  • Chromatin was prepared from nuclear lysate of the human MCF7 breast epithelial adenocarcinoma cells. The cross-linking (X-ChiP) technique was used, crosslinking was performed for 15 minutes in formaldehyde. The primary antibody was used in concentration of 0.2 µg/µg chromatin and incubated with the sample for 16 hours at 4°C in SDS, DOC, TritonX-100, EDTA, HEPES, NaCl. The immunoprecipitated DNA was quantified by real time PCR. Ct values were converted to DNA copy numbers using a standard curve in the Q-PCR step. The number of binding events detected for each test reaction was then calculated by taking into account the DNA copy number, cell equivalents of chromatin used in the ChIP and PCR, and primer pair amplification efficiency.

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab817 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab817, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • IHC - Wholemount of Caenorhabditis elegans embryo labelling RNA polymerase II CTD repeat YSPTSPS with ab817. Sample was incubated with primary antibody (1/100 in PBS + 3% BSA + 0.1% Triton-X 100) for 24 hours at 4°C. ab150113, an Alexa Flour® 488-conjugated goat anti-mouse IgG polyclonal (undiluted) was used as the secondary antibody.

    See Abreview

References for Anti-RNA polymerase II CTD repeat YSPTSPS antibody [8WG16] - ChIP Grade (ab817)

This product has been referenced in:
  • Lei M  et al. Arabidopsis EDM2 promotes IBM1 distal polyadenylation and regulates genome DNA methylation patterns. Proc Natl Acad Sci U S A 111:527-32 (2014). Read more (PubMed: 24248388) »
  • Shin MR  et al. Sense transgene-induced post-transcriptional gene silencing in tobacco compromises the splicing of endogenous counterpart genes. PLoS One 9:e87869 (2014). ChIP . Read more (PubMed: 24586294) »

See all 77 Publications for this product

Product Wall

Application Immunocytochemistry/ Immunofluorescence
Blocking step (agent) for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Sample Quail Cell (Fibroblast QT6 cells)
Specification Fibroblast QT6 cells
Permeabilization Yes - 0.1% Triton
Fixative Paraformaldehyde
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Submitted Jul 31 2014

Application IHC - Wholemount
Sample Caenorhabditis elegans Embryo (C. elegans embryo)
Specification C. elegans embryo
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Submitted Mar 18 2014

Application IHC - Wholemount
Sample Fruit fly (Drosophila melanogaster) Tissue (Giant polytene chromosome in salivary glands)
Specification Giant polytene chromosome in salivary glands
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Submitted Dec 27 2013

Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Denaturing (6%)
Sample Caenorhabditis elegans Tissue lysate - nuclear (C. elegans whole body)
Specification C. elegans whole body
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Dec 25 2013

Application Western blot
Loading amount 6 µg
Gel Running Conditions Reduced Denaturing (6%)
Sample Mouse Tissue lysate - nuclear (Mouse embryonic fibroblasts)
Specification Mouse embryonic fibroblasts
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Dec 19 2013

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: 23°C
Sample Mouse Cell (Mouse spermatocyte)
Specification Mouse spermatocyte
Permeabilization Yes - 0.3% Triton X-100
Fixative Paraformaldehyde
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Submitted Dec 19 2013

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Sample Human Cell (HEK293T cells)
Specification HEK293T cells
Permeabilization Yes - 0.3% Triton X-100
Fixative Paraformaldehyde
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Submitted Dec 19 2013

Application Immunocytochemistry
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Sample Fruit fly (Drosophila melanogaster) Cell (imaginal discs)
Specification imaginal discs
Permeabilization Yes - 0.1% tritonX-100
Fixative Formaldehyde
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Dr. Yoshihiro Inoue

Verified customer

Submitted Dec 12 2013

Abcam has not validated the combination of species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Permeabilization Yes - 0.5% Triton X100 in PBS
Fixative Paraformaldehyde
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Dr. Kirk McManus

Verified customer

Submitted Aug 29 2013

The earliest reference I found for the clone is:

J Biol Chem. 1989 Jul 5;264(19):11511-20.
PMID:2472398
http://www.jbc.org/content/264/19/11511.long

It describes production of the clone and some characterization but as f...

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