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

概述

  • 产品名称Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5)抗体- ChIP Grade
    参阅全部 RNA polymerase II CTD repeat YSPTSPS 一抗
  • 描述
    兔多克隆抗体to RNA polymerase II CTD repeat YSPTSPS (phospho S5) - ChIP Grade
  • 特异性This antibody recognises the phosphorylated serine found in the amino acid 5 position of the C-terminal domain repeat YSPTSPS of RNA polymerase II.
  • 经测试应用适用于: IP, IHC-P, ELISA, IHC - Wholemount, IHC-Fr, CHIPseq, WB, ChIP, ICC/IFmore details
  • 种属反应性
    与反应: Mouse, Rat, Human, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Zebrafish
    预测可用于: a wide range of other species
  • 免疫原

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

    (Peptide available as ab18488.)

  • 阳性对照
    • 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.
  • 常规说明

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

性能

应用

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.

应用 Ab评论 说明
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.
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.

靶标

  • 功能DNA-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 an 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.
  • 序列相似性Belongs to the RNA polymerase beta' chain family.
  • 结构域The C-terminal domain (CTD) serves as a platform for assembly of factors that regulate transcription initiation, elongation, termination and mRNA processing.
  • 翻译后修饰The tandem heptapeptide 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 heptapeptide 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.
    Among tandem heptapeptide repeats of the C-terminal domain (CTD) some do not match the Y-S-P-T-S-P-S consensus, the seventh serine residue 'Ser-7' being replaced by a lysine. 'Lys-7' in these non-consensus heptapeptide repeats can be alternatively acetylated, methylated and dimethylated. EP300 is one of the enzyme able to acetylate 'Lys-7'. Acetylation at 'Lys-7' of non-consensus heptapeptide repeats is associated with 'Ser-2' phosphorylation and active transcription. It may regulate initiation or early elongation steps of transcription specially for inducible genes.
    Methylated at Arg-1810 prior to transcription initiation when the CTD is hypophosphorylated, phosphorylation at Ser-1805 and Ser-1808 preventing this methylation. Symmetrically or asymmetrically dimethylated at Arg-1810 by PRMT5 and CARM1 respectively. Symmetric or asymmetric dimethylation modulates interactions with CTD-binding proteins like SMN1/SMN2 and TDRD3. SMN1/SMN2 interacts preferentially with the symmetrically dimethylated form while TDRD3 interacts with the asymmetric form. Through the recruitment of SMN1/SMN2, symmetric dimethylation is required for resolving RNA-DNA hybrids created by RNA polymerase II, that form R-loop in transcription terminal regions, an important step in proper transcription termination. CTD dimethylation may also facilitate the expression of select RNAs. Among tandem heptapeptide repeats of the C-terminal domain (CTD) some do not match the Y-S-P-T-S-P-S consensus, the seventh serine residue 'Ser-7' being replaced by a lysine. 'Lys-7' in these non-consensus heptapeptide repeats can be alternatively acetylated, methylated and dimethylated. Methylation occurs in the earliest transcription stages and precedes or is concomitant to 'Ser-5' and 'Ser-7' phosphorylation.
    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.
  • 细胞定位Nucleus.
  • Information by UniProt
  • 数据库链接
    see all
  • 别名
    • DNA directed RNA polymerase II A 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 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 图像

  • 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 HRP (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).

  • 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

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  • 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
  • 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 minute
  • (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
  • 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, a goat anti-rabbit Alexa 488 - (1/1000), was used as the secondary antibody.

    See Abreview

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

This product has been referenced in:
  • Magbanua JP  et al. A variably occupied CTCF binding site in the ultrabithorax gene in the Drosophila bithorax complex. Mol Cell Biol 35:318-30 (2015). Read more (PubMed: 25368383) »
  • Bazot Q  et al. Epstein-Barr Virus Proteins EBNA3A and EBNA3C Together Induce Expression of the Oncogenic MicroRNA Cluster miR-221/miR-222 and Ablate Expression of Its Target p57KIP2. PLoS Pathog 11:e1005031 (2015). Read more (PubMed: 26153983) »

See all 89 Publications for this product

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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...

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Application Western blot
Sample Human Cell lysate - whole cell (U2OS cells)
Gel Running Conditions Reduced Denaturing (4-12% Gradient Gel)
Loading amount 20 µg
Specification U2OS cells
Blocking step Licor Blocking Buffer as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C
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提交于 Dec 01 2016

Application Western blot
Loading amount 1e+006 cells
Gel Running Conditions Reduced Denaturing (12%)
Sample Human Cell lysate - whole cell (Breast)
Specification Breast
Treatment tnf-a
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
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提交于 Jan 07 2015

Application ChIP
Detection step Real-time PCR
Sample Mouse Cell lysate - whole cell (Colon)
Specification Colon
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde 4%
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提交于 Dec 17 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (6%)
Sample Human Cell lysate - whole cell (U2OS cells)
Specification U2OS cells
Treatment 1 ´M CPT
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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提交于 Nov 25 2014

Application ChIP
Detection step Real-time PCR
Sample Human Cell lysate - whole cell (Breast cell line- MCF10A)
Specification Breast cell line- MCF10A
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 Cells were treated with TNF- alpha, and transcription of IL8 was tested.
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提交于 Nov 04 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
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提交于 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
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提交于 Dec 30 2013

Application IHC - Wholemount
Sample Caenorhabditis elegans Embryo (C. elegans larvae)
Specification C. elegans larvae
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提交于 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
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提交于 Dec 30 2013

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