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

Overview

• Product nameAnti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP GradeSee all RNA polymerase II CTD repeat YSPTSPS primary antibodies ...
• Description
  Rabbit polyclonal to RNA polymerase II CTD repeat YSPTSPS - ChIP Grade
• Tested applicationsWB, IP, ICC/IF, ChIP more details
• Species reactivity
  Reacts with: Mouse, Human
  
• Immunogen

  Synthetic peptide of Human RNA polymerase II CTD repeat YSPTSPS.

  Read Abcam's proprietary immunogen policy

  (Peptide available as ab17564.)

• Positive controlThis antibody gave a positive control in both HeLa and HEK293 whole cell lysates.

Properties

• FormLiquid
• Storage instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
• Storage bufferPreservative: 0.02% Sodium Azide
  Constituents: 1% BSA, PBS, pH 7.4
• Concentration information loading...
• PurityImmunogen affinity purified
• Clonality Polyclonal
• IsotypeIgG
• Research Areas
  • Epigenetics and Nuclear Signaling
  • Transcription
  • Polymerase associated factors
  • Pol II Transcription
  • Polymerase

  • Epigenetics and Nuclear Signaling
  • Transcription
  • RNA polymerase

  • Epigenetics and Nuclear Signaling
  • ChIP'ing antibodies
  • ChIP'ing antibodies

Applications

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
  modificationsThe 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.

Target information above from: UniProt accession P24928 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
• Database links
  • Entrez Gene: 5430 Human
  • Entrez Gene: 20020 Mouse
  • Omim: 180660 Human
  • SwissProt: P24928 Human
  • SwissProt: P08775 Mouse
  • Unigene: 270017 Human
  • Unigene: 16533 Mouse

• Alternative names
  DNA directed RNA polymerase II A antibodyDNA-directed RNA polymerase II largest subunit antibodyDNA-directed RNA polymerase II largest subunit RNA polymerase II 220 kd subunit antibody

  DNA-directed RNA polymerase II subunit A antibodyDNA-directed RNA polymerase II subunit RPB1 antibodyDNA-directed RNA polymerase III largest subunit antibodyhRPB220 antibodyhsRPB1 antibodyPOLR2 antibodyPolr2a antibodyPOLRA antibodyPolymerase (RNA) II (DNA directed) polypeptide A 220kDa antibodyPolymerase (RNA) II (DNA directed) polypeptide A antibodyRNA pol II CTD antibodyRNA polymerase II subunit B1 antibodyRNA-directed RNA polymerase II subunit RPB1 antibodyRPB1 antibodyRPB1_HUMAN antibodyRPBh1 antibodyRpIILS antibodyRPO2 antibodyRPOL2 antibody

  see all

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

• 

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

  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.
     

• 

  Immunocytochemistry/ Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)Image courtesy of an abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada
  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

• 

  Immunocytochemistry/ Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)

  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.

• 

  Western blot - RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)
  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 RNA polymerase II CTD repeat YSPTSPS peptide (ab17564) at 1 µg/ml
  Lane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide (ab17564) at 1 µg/ml
  Lane 5 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488) at 1 µg/ml
  Lane 6 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488) at 1 µg/ml
  Lane 7 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (ab12793) at 1 µg/ml
  Lane 8 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (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
• 

  Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)
  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 70^oC; 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.