Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2)抗体- ChIP Grade (ab5095)

概述

  • 产品名称Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2)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 S2) - ChIP Grade
  • 特异性This antibody recognises the phosphorylated serine found in the amino acid 2 position of the C-terminal domain repeat YSPTSPS.
  • 经测试应用适用于: ELISA, IHC-FoFr, ChIP, ChIP/Chip, IHC-P, IHC-Fr, ICC/IF, IHC - Wholemount, Dot blot, WB, CHIPseqmore details
  • 种属反应性
    与反应: Mouse, Rat, Human, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe
    预测可用于: a wide range of other species
  • 免疫原

    Synthetic peptide conjugated to KLH derived from within residues 1600 - 1700 of Saccharomyces cerevisiae RNA polymerase II CTD repeat YSPTSPS, phosphorylated at S2.

    (Peptide available as ab12793.)

  • 阳性对照
    • This antibody gave a positive signal in Hela Whole Cell Lysate and S.cerevisiae extract. IHC-P: Human pancreas and tonsil tissue, mouse and rat kidney tissue.

性能

应用

Our Abpromise guarantee covers the use of ab5095 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
ELISA 1/438.
IHC-FoFr Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration.
ChIP/Chip Use at an assay dependent concentration.
IHC-P 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
IHC-Fr Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
IHC - Wholemount Use at an assay dependent concentration.
Dot blot 1/3000. AbReview 31067
WB Use a concentration of 1 µg/ml. Detects a band of approximately 240 kDa (predicted molecular weight: 217 kDa).Can be blocked with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (ab12793).
CHIPseq Use 2-0.3 µg for µg of chromatin.

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 S2) antibody - ChIP Grade 图像

  • All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : S.cerevisiae (Y190) Whole Cell Lysate
    Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell 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) Whole Cell Lysate with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (ab12793) at 1 µg/ml
    Lane 6 : S.cerevisiae (Y190) Whole Cell Lysate with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (ab12793) at 1 µg/ml
    Lane 7 : 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 8 : S.cerevisiae (Y190) Whole Cell Lysate with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (ab18488) at 1 µg/ml

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size : 217 kDa
    Observed band size : 240 kDa (why is the actual band size different from the predicted?)
  • Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab5095 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Weak nuclear staining on epithelium cells and glomerulus cells of rat kidney was observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

    Perform heat mediated antigen retrieval using Tris/EDTA buffer, pH9.

  • ab5095 at 4ug/ml in ChIP of RAW macrophages. Nuclear cell lysate of mouse RAW macrophages (expressing c-fms) were formaldehyde cross linked and ChIP tested with ab5095. The nuclear preparation was frozen before sonication with a probe sonicator. All buffers used contained protease inhibitors. 3T3 fibroblasts (not expressing c-fms) were used as the negative control.

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  • 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 1/500 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
  • Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab5095 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on epithelium cells and glomerulus cells of mouse kidney was observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

    Perform heat mediated antigen retrieval using Tris/EDTA buffer, pH9.

  • Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab5095 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human tonsil was observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

    Perform heat mediated antigen retrieval using Tris/EDTA buffer, pH9.

  • Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab5095 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on epithelium cells and pancreas islet cells of human pancreas was observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

    Perform heat mediated antigen retrieval using Tris/EDTA buffer, pH9.

  • Diluted ab5095 was bound to immobilised phospho- or control peptides (1 microgram per mL). The antibody was detected by goat anti-rabbit IgG (HRP) (ab97080; diluted 50000 times), and signal was developed by TMB substrate.

  • IHC - Wholemount of Caenorhabditis elegans larvae labelling RNA polymerase II CTD repeat YSPTSPS (phospho S2) with ab5095. 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 goat anti-rabbit Alexa Fluor® 488 (1/1000), was used as the secondary antibody.

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  • Image courtesy of Human Protein Atlas

    ab5095 staining in human brain, showing staining of the Purkinje cells (in brown). Paraffin embedded brain tissue was incubated with ab5095 (1:900 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. ab5095 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

    .

  • ICC/IF image of ab5095 stained MCF7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab5095, 1µg/ml) overnight at +4°C. The secondary antibody (green) was a goat anti-rabbit DyLight® 488 (IgG - H&L, pre-adsorbed) (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095)参考文献

This product has been referenced in:
  • Rother S  et al. NF-?B-repressing factor phosphorylation regulates transcription elongation via its interactions with 5'?3' exoribonuclease 2 and negative elongation factor. FASEB J 30:174-85 (2016). Read more (PubMed: 26340924) »
  • Ard R & Allshire RC Transcription-coupled changes to chromatin underpin gene silencing by transcriptional interference. Nucleic Acids Res N/A:N/A (2016). Read more (PubMed: 27613421) »

See all 91 Publications for this product

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Abcam has not validated the combination of species/application used in this Abreview.
Application Western blot
Sample Dog Cell lysate - whole cell (Canine Kidney Epithelial MDCK cells)
Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
Loading amount 50000 cells
Specification Canine Kidney Epithelial MDCK cells
Blocking step Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
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Abcam has not validated the combination of species/application used in this Abreview.
Application Western blot
Sample Rabbit Cell lysate - whole cell (Rabbit Reticulocyte)
Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
Loading amount 0.5 µg
Specification Rabbit Reticulocyte
Blocking step Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
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提交于 Nov 21 2016

Application Immunoprecipitation
Sample Human Cell lysate - whole cell (A549 (human lung epithelium cells))
Total protein in input 1e+007 cells
Immuno-precipitation step Other - Protein G Dynabeads
Specification A549 (human lung epithelium cells)
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提交于 Jul 07 2016

Abcam has not validated the combination of species/application used in this Abreview.
Application Western blot
Sample Hamster Cell lysate - whole cell (BHK-21 (Kidney Fibroblasts))
Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
Loading amount 50000 cells
Specification BHK-21 (Kidney Fibroblasts)
Blocking step Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
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提交于 Jun 19 2016

Application Western blot
Sample Mouse Cell lysate - whole cell (Mouse embryonic fibroblasts (MEFs))
Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gradient gel)
Loading amount 50000 cells
Specification Mouse embryonic fibroblasts (MEFs)
Blocking step Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
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提交于 Feb 19 2016

Application Western blot
Sample Human Cell lysate - whole cell (Human Foreskin Fibroblasts)
Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gradient gel)
Loading amount 50000 cells
Specification Human Foreskin Fibroblasts
Blocking step Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
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提交于 Feb 10 2016

Application Western blot
Sample Human cytomegalovirus Cell lysate - whole cell (HCMV-infected human foreskin fibroblasts)
Gel Running Conditions Reduced Denaturing (12.5% gel)
Loading amount 500000 cells
Treatment 1 µM Shield-1
Specification HCMV-infected human foreskin fibroblasts
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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提交于 Jun 16 2015

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 10 2014

Application ChIP
Detection step Real-time PCR
Sample Human Cell lysate - whole cell (Breast- MCF10A)
Specification Breast- 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

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