Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S) antibody [4H8] - ChIP Grade (ab5408)

Overview

• Product nameAnti-RNA polymerase II CTD repeat YSPTSPS (phospho S) antibody [4H8] - ChIP GradeSee all RNA polymerase II CTD repeat YSPTSPS primary antibodies ...
• Description
  Mouse monoclonal [4H8] to RNA polymerase II CTD repeat YSPTSPS (phospho S) - ChIP Grade
• SpecificityELISA and peptide blocking show that the specificity of ab5408 towards the different phospho-forms varies between batches, with a preference towards the phosphoS5 form. ab26721 is another suitable antibody that shows a greater preference for unmodified RNAPII in WB peptide inhibition.
• Tested applicationsFlow Cyt, ICC/IF, ChIP, ChIP/Chip, WB, ELISA, IP, CHIPseq more details
• Species reactivity
  Reacts with: Mouse, Human, Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, African Green Monkey
  Predicted to work with: Hamster
• Immunogen

  Synthetic peptide: YSPTSpPS (Human). The sequence is repeated multiple times in the C-terminal domain of RNA polymerase II.

• Positive controlThis antibody gave a positive signal in methanol fixed/Tween permeabilised HeLa cells within Flow Cytometry.

Properties

• FormLiquid
• Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
• Storage bufferpH: 7.50
  Preservative: 0.02% Sodium azide
  Constituent: PBS
  Note: Contains 0.4M Arginine
• Concentration information loading...
• PurityIgG fraction
• Clonality Monoclonal
• Clone number4H8
• MyelomaSp2/0-Ag14
• IsotypeIgG1
• 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: 177190 Caenorhabditis elegans
  • Entrez Gene: 32100 Fruit fly (Drosophila melanogaster)
  • Entrez Gene: 5430 Human
  • Entrez Gene: 20020 Mouse
  • Entrez Gene: 851415 Saccharomyces cerevisiae
  • Omim: 180660 Human
  • SwissProt: P18616 Arabidopsis thaliana
  • SwissProt: P16356 Caenorhabditis elegans

  • SwissProt: P04052 Fruit fly (Drosophila melanogaster)
  • SwissProt: P24928 Human
  • SwissProt: P08775 Mouse
  • Unigene: 2925 Fruit fly (Drosophila melanogaster)
  • Unigene: 270017 Human
  • Unigene: 16533 Mouse

  see all

• 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 (phospho S) antibody [4H8] - ChIP Grade images

• 

  ChIP - RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)

  Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab5408 (blue), and 20 µl of protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

• 

  Western blot - RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)
  All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S) antibody [4H8] - ChIP Grade (ab5408) at 1 µg/ml
  
  Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
  Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide (ab17564) at 1 µg/ml
  Lane 3 : 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 4 : HeLa (Human epithelial carcinoma 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/ml per lane.
  
  Secondary
  Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution
  
  Performed under reducing conditions.
  
  Predicted band size : 217 kDa
  Observed band size : 260 kDa (why is the actual band size different from the predicted?)
  
  
  Exposure time : 30 seconds
• 

  Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)Michael Mancini, Baylor College of Medicine
  HeLa or MCF7 cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/1000 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-mouse Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minutes in TBS-T. Coverslips were then counterstained with DAPI in TBS-T for 1-2 minutes, TBS-T was then added and the coverslips mounted. Red indicates staining by ab5408, blue by DAPI.
• 

  Western blot - RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)
  
  
  Predicted band size : 217 kDa
  
  
  

  Lane 1: buffer,

  Lane 2: extract from human 293T cells as positive control,

  Lanes 3 and 4: egg extracts made from wild type N2 worms and worms grown on RNAi food against dicer,

  Lanes 5-8: intact worms in mentioned larval stage and eggs boiled directly in SDS-PAGE buffer and loaded

  This image was kindly supplied as part of the review submitted by Shveta Bagga.

• 

  ELISA - RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)
  ELISA using ab5408 at varying antibody concentrations. Curve_SPL4 indicates binding to RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488). Binding to the following peptides was much weaker: Curve_SPL5 RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (ab12793), Curve_SPL6 RNA polymerase II CTD repeat YSPTSPS peptide (ab12795).
• 

  Flow Cytometry-Anti-RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade(ab5408)
  Overlay histogram showing HeLa cells stained with ab5408 (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 (ab5408, 1µg/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 IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.