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 - Wholemount, Flow Cyt, IHC-Fr, CHIPseq, WB, ChIP, ICC/IF more details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Saccharomyces cerevisiae, Xenopus laevis, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Zebrafish
    Predicted to work with: a wide range of other species
  • Immunogen

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

    (Peptide available as ab18488.)

  • Positive control
    • This 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

Applications

Our Abpromise guarantee covers the use of ab5131 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
IP 1/50.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ELISA 1/438.
IHC - Wholemount Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
CHIPseq Use at an assay dependent concentration.
WB 1/1000. Detects a band of approximately 240 kDa (predicted molecular weight: 217 kDa).Can be blocked with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488).
ChIP Use 2-25 µg for µg of chromatin.
ICC/IF Use a concentration of 1 µg/ml.

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.
  • 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
    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 (phospho S5) antibody - ChIP Grade images

  • IHC - Wholemount of Caenorhabditis elegans larvae labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab5131. The sample was incubated with primary antibody (1/500 in PBS + 3% BSA + 0.1% Triton X-100) for 12 hours at 4°C. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal (1/1000), was used as the secondary antibody.

    See Abreview

  • All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (ab5131) at 1/5000 dilution

    Lane 1 : Xenopus laevis whole tissue lysate treated with DMSO for 24 hours
    Lane 2 : Xenopus laevis whole tissue lysate treated with CDK inhibitor for 24 hours

    Secondary
    HRP-conjugated goat anti-rabbit IgG polyclonal at 1/10000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 217 kDa
    Observed band size : 240 kDa (why is the actual band size different from the predicted?)


    Exposure time : 1 minute

    This image is courtesy of an anonymous Abreview

    See Abreview

  • 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 S. cerevisiae RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml
    Lane 4 : S.cerevisiae (Y190) Whole Cell Lysate with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml
    Lane 5 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488) at 1 µg/ml
    Lane 6 : S.cerevisiae (Y190) 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) Nuclear Lysate with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (ab12793) at 1 µg/ml
    Lane 8 : S.cerevisiae (Y190) 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) at 1/3000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 217 kDa


    Exposure time : 30 seconds
  • 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.

  • 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 Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488) at 1 µg/ml
    Lane 4 : HeLa nuclear extract with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS peptide (ab12795) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-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?)

    Western blot using ab5131.

    Lane 1: ab5131 at 1/500
    Lane 2: ab5131 at 1/2000
    Lane 3: ab5131 at 1/500 blocked with phospho peptide
    Lane 4: ab5131 at 1/500 blocked with non-phospho control peptide

    Phospho peptide is YSPTSpPSYSPTSpPS-GGC (ab18488)
    Non-phospho control peptide is YSPTSPSYSPTSPS-GGC (ab12795)

    Secondary ab: Goat anti-rabbit IgG ab6721 (1/5000)
    Lanes 1 to 4: 20µg of HeLa nuclear extract per lane
    Blocking peptides used at 1µg/ml.
    Exposure time: 30 seconds
    Expected molecular weight: ~240 kDa

  • 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).

  • (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
  • 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 minuteHeLa 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.

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

This product has been referenced in:
  • Eun B  et al. Promoter cross-talk via a shared enhancer explains paternally biased expression of Nctc1 at the Igf2/H19/Nctc1 imprinted locus. Nucleic Acids Res 41:817-26 (2013). IP ; Human . Read more (PubMed: 23221643) »
  • Khan P  et al. Mitogen- and stress-activated protein kinases 1 and 2 are required for maximal trefoil factor 1 induction. PLoS One 8:e63189 (2013). ChIP ; Human . Read more (PubMed: 23675462) »

See all 52 Publications for this product

Product Wall

Thank you for your patience.

The lab have not tested whether the antibody will detect phospho S5 if S2 is also phosphorylated. The peptides available contain either phospho S2 or phospho S5, not both modifications, therefore we check for cro...

Read More
Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - 0.1% Triton in PBS
Sample Human Cell (MCF-7 (invasive ductal carcinoma line))
Specification MCF-7 (invasive ductal carcinoma line)
Gating Strategy isotype control (black line in histogram)
Preparation Cell harvesting/tissue preparation method: accutase and accumax dissociation
Sample buffer: PBS 7.4 + 10% FBS for washing
Username

Abcam user community

Verified customer

Submitted Mar 11 2014

Application Immunohistochemistry (PFA perfusion fixed frozen sections)
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 23°C
Antigen retrieval step None
Sample Rat Tissue sections (Rat placenta tissue)
Specification Rat placenta tissue
Permeabilization Yes - 0.3% Triton X-100
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Dec 30 2013

Application Immunocytochemistry
Blocking step Serum as blocking agent for 20 minute(s) · Concentration: 3% · Temperature: 23°C
Sample Human Cell (Human embryonic stem cells)
Specification Human embryonic stem cells
Permeabilization Yes - 0.3% Triton X-100
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Dec 30 2013

Application IHC - Wholemount
Sample Caenorhabditis elegans Embryo (C. elegans larvae)
Specification C. elegans larvae
Username

Abcam user community

Verified customer

Submitted Dec 30 2013

Application IHC - Wholemount
Sample Fruit fly (Drosophila melanogaster) Tissue (giant polytene chromosome in salivary glands)
Specification giant polytene chromosome in salivary glands
Username

Abcam user community

Verified customer

Submitted Dec 30 2013

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing (6%)
Sample Xenopus laevis Tissue lysate - whole (Xenopus embryo)
Specification Xenopus embryo
Treatment 25 ´M DMSO or CDK inhibitor for 24hrs
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Dec 30 2013

Application Immunocytochemistry
Blocking step BSA as blocking agent for 20 minute(s) · Concentration: 3% · Temperature: 23°C
Sample Fruit fly (Drosophila melanogaster) Cell (intestinal stem cells)
Specification intestinal stem cells
Permeabilization Yes - 0.3% Triton X-100
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Dec 27 2013

The input is the sample that is used to produce the standard curve for q-PCR. It's essentially chromatin that has not been immunoprecipiated using a sample antibody. Out test samples are normalised against the standard curve and this allows us to get...

Read More
Application ELISA
Sample Human Recombinant protein (YSPTSPS (CTD repeat) peptide)
Specification YSPTSPS (CTD repeat) peptide
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 0.1% · Temperature: 22°C
Type Other
Username

Abcam user community

Verified customer

Submitted Feb 20 2013

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