Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)

Overview

• Product nameAnti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP GradeSee all RNA polymerase II CTD repeat YSPTSPS primary antibodies ...
• Description
  Rabbit polyclonal to RNA polymerase II CTD repeat YSPTSPS (phospho S5) - ChIP Grade
• SpecificityThis antibody recognises the phosphorylated serine found in the amino acid 5 position of the C-terminal domain repeat YSPTSPS of RNA polymerase II.
• Tested applicationsIP, IHC-P, ELISA, IHC-Fr, CHIPseq, WB, ChIP, ICC/IF more details
• Species reactivity
  Reacts with: Mouse, Rat, Human, Saccharomyces cerevisiae, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Zebrafish
  Predicted to work with: Caenorhabditis elegans, a wide range of other species
• Immunogen

  Synthetic peptide of Saccharomyces cerevisiae RNA polymerase II CTD repeat YSPTSPS, phosphorylated at S5.

  Read Abcam's proprietary immunogen policy

  (Peptide available as ab18488.)

• Positive controlThis antibody gave a positive signal in S.cerevisiae (Y190) Whole Cell Lysate and HeLa Nuclear Lysate within Western blot, HeLa whole cell lysate within Immunofluorescence and Human urinary bladder tissue within Immunohistochemistry.
• General notes

  
  

  Phosphorylation of RNA polymerase II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism.

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
• Primary antibody notes Phosphorylation of RNA polymerase II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism.
• 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: 177190 Caenorhabditis elegans
  • Entrez Gene: 32100 Fruit fly (Drosophila melanogaster)
  • Entrez Gene: 5430 Human
  • Entrez Gene: 20020 Mouse
  • Entrez Gene: 363633 Rat
  • Entrez Gene: 851415 Saccharomyces cerevisiae
  • Omim: 180660 Human
  • 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 S5) antibody - ChIP Grade images

• 

  Western blot - RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)
  All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131) at 1 µg/ml
  
  Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
  Lane 2 : S.cerevisiae (Y190) Whole Cell Lysate
  Lane 3 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate with RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml
  Lane 4 : S.cerevisiae (Y190) Whole Cell Lysate with RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml
  Lane 5 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488) at 1 µg/ml
  Lane 6 : S.cerevisiae (Y190) 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) Nuclear Lysate with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (ab12793) at 1 µg/ml
  Lane 8 : S.cerevisiae (Y190) 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) at 1/3000 dilution
  developed using the ECL technique
  
  Performed under reducing conditions.
  
  Predicted band size : 217 kDa
  
  
  Exposure time : 30 seconds
• 

  ChIP - RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)

  Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 mins. The  ChIP was performed with 25 µg of chromatin, 2 µg of  ab5131 (blue), and 20 µl 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 y-Actin gene (active). Schematic diagram of the y-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.

• 

  Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)
  Lanes 1, 3 & 4 : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131) at 1/500 dilution
  Lane 2 : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131) at 1/2000 dilution
  
  Lane 1 : HeLa nuclear extract
  Lane 2 : HeLa nuclear extract
  Lane 3 : HeLa nuclear extract with RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (ab18488) at 1 µg/ml
  Lane 4 : HeLa nuclear extract with RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml
  
  Lysates/proteins at 20 µg per lane.
  
  Secondary
  Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution
  
  Performed under reducing conditions.
  
  Predicted band size : 217 kDa
  Observed band size : 250 kDa (why is the actual band size different from the predicted?)
  
• 

  Immunocytochemistry/ Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)
  ICC/IF image of ab5131 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab5131, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and 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).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131)
  (Image courtesy of Human Protein Atlas) ab5131 staining RNA Polymerase in Human urinary bladder tissue. Paraffin embedded human skin tissue was incubated with ab5131 for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. ab5131 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org
• 

  Immunofluorescence - RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (ab5131)Michael Mancini, Baylor College of Medicine
  HeLa 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 1/160 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-rabbit 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 ab5131, blue by DAPI.

Protocols

• Abreview protocol for ChIP using ab5131