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, CHIPseqmore details
  • Species reactivity
    Reacts with: Mouse, Rat, 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 control
    • This antibody gave a positive signal in methanol fixed/Tween permeabilised HeLa cells within Flow Cytometry.

Properties

Applications

Our Abpromise guarantee covers the use of ab5408 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
Flow Cyt Use 1µg for 106 cells.
ICC/IF 1/1000.
ChIP Use 1-4µg for 106 cells.
ChIP/Chip Use at an assay dependent concentration. PubMed: 19703992
WB Use a concentration of 1 µg/ml. Detects a band of approximately 260 kDa (predicted molecular weight: 217 kDa).
ELISA Use at an assay dependent concentration.
IP Use at an assay dependent concentration. See Abreviews.
CHIPseq Use 2-0.3 µg for µg of chromatin.

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

  • 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.

  • 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 Human RNA polymerase II CTD repeat YSPTSPS peptide (ab17564) at 1 µg/ml
    Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488) at 1 µg/ml
    Lane 4 : HeLa (Human epithelial carcinoma cell line) 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/ml per lane.

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (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
  • 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.


  • 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 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).
  • 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.

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

This product has been referenced in:
  • Hemsley PA  et al. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes. Plant Cell 26:465-84 (2014). ChIP ; Arabidopsis thaliana . Read more (PubMed: 24415770) »
  • Zhang Y  et al. Canonical nucleosome organization at promoters forms during genome activation. Genome Res 24:260-6 (2014). Zebrafish . Read more (PubMed: 24285721) »

See all 74 Publications for this product

Product Wall

Application ChIP
Detection step Real-time PCR
Sample Human Cell lysate - whole cell (HeLa Cells)
Specification HeLa Cells
Negative control Uninduced Sample
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% Formaldehyde
Positive control Induced Sample
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Submitted Jul 21 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
Sample African Green Monkey Cell (COS7)
Specification COS7
Permeabilization Yes - Triton X100 0.3%
Fixative Paraformaldehyde
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Submitted Jul 09 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Mouse Cell lysate - nuclear (CH12 and MEL cell lines)
Specification CH12 and MEL cell lines
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Submitted Mar 14 2014

Application ChIP
Detection step Real-time PCR
Sample Human Cell lysate - nuclear (Teratocarcinoma)
Specification Teratocarcinoma
Negative control Non-transcribed gene
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde
Positive control GAPDH 1kb downstream the TSS
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Submitted Jul 02 2013

Thank you for contacting us. We do have the primers/probes for the figure for ab5408 on our catalogue :)

GAPDH: ab85378
RPL30: ab85062
ALDOA: ab85773
MYO-D: ab85776
SERPINA: ab85777

Please let me know if I can be of fu...

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Merci de nous avoir contactés.

Le numéro de commande pour ab26721 et ab1791 est xxxxxxxx. Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

Le numéro de com...

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Merci de nous avoir contactés.

Nous sommes désolés d'apprendre que les produits que vous avez reçus ne fonctionnent pas comme attendu. Je me permets de vous transférer le numéro de commande des rempl...

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Merci de nous avoir contactés.

Nous sommes désolés d'apprendre que les produits que vous avez reçus ne fonctionnent pas comme attendu. Je suis d’accord pour vous envoyer ab26721, ab106293 et ab1791 en rempla...

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Merci de nous avoir contactés.

J’ai eu ma réponse de nos laboratoires et ce cas et assez compliqué. Premièrement je tiens à vous remercier d’avoir patienté et j’apprécie &eacu...

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Thank you for contacting us.

ab5408 is part of the RNA polymerase II CTD Panel reference ab103968 which is composed of:

25µg of anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (http://www.abcam.com/a...

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