Anti-RNA polymerase II CTD repeat YSPTSPS抗体[8WG16] - ChIP Grade (ab817)

概述

  • 产品名称Anti-RNA polymerase II CTD repeat YSPTSPS抗体[8WG16] - ChIP Grade
    参阅全部 RNA polymerase II CTD repeat YSPTSPS 一抗
  • 描述
    小鼠单克隆抗体[8WG16] to RNA polymerase II CTD repeat YSPTSPS - ChIP Grade
  • 特异性Clone 8WG16 recognizes YSPTSPS in the C-terminal domain. The antibody cross-reacts with wheat, yeast, mouse, c. elegans, x. laevis, and most eukaryotic RNAPII.
  • 经测试应用适用于: CHIPseq, WB, ICC/IF, IHC - Wholemount, ChIP, Flow Cyt, IPmore details
  • 种属反应性
    与反应: Mouse, Cow, Human, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Quail, Neurospora crassa , Rice
    预测可用于: a wide range of other species
  • 免疫原

    Other Immunogen Type corresponding to RNA polymerase II CTD repeat YSPTSPS. Immunogen: Wheat germ RNA Polymerase II

  • 常规说明This antibody may be used to detect unproteolyzed RNA polymerase II or to inhibit specific transcription from class II promoters. This is a polyol-responsive antibody and can be used in gentle purifications of RNA polymerase II from from wheat germ, calf thymus, and yeast and is likely to purify RNA polymerase II from most eukaryotic organisms. It inhibits promoter-directed transcription, but it does not inhibit elongation in the nonspecific transcription assay.

性能

应用

Our Abpromise guarantee covers the use of ab817 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
CHIPseq Use at an assay dependent concentration. PubMed: 19251593Use 2ug per 0.3ml of sonicated chromatin.
WB Use a concentration of 0.25 - 2 µg/ml. Detects a band of approximately 217 kDa (predicted molecular weight: 217 kDa).
ICC/IF Use a concentration of 1 - 5 µg/ml.
IHC - Wholemount Use at an assay dependent concentration.

See Abreview.

ChIP Use at an assay dependent concentration.

See Abreview; the user recommends using anti-mouse IgG coated Dynabeads instead of Protein A to recover the precipitate).

Flow Cyt Use 0.5µg for 106 cells. ab170191-Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.
IP Use at an assay dependent concentration.

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 antibody [8WG16] - ChIP Grade 图像

  • Various regions across the Actin2/7 loci were tested for the presence of RNA polymerase II CTD repeat YSPTSPS. A nuclear lysate from Arabidopsis thaliana seedlings was crosslinked using 1% formaldehyde for 30 seconds.  The ChIP was performed with 0.1 µg of ab817 per µg of chromatin; incubated together for 16 hours at 4°C.  The immunoprecipitated DNA was quantified by Real-Time PCR.  The bottom panel indicates the positive (ab817) and negative controls (no antibody) at region B3.

    See Abreview

  • Chromatin was prepared from nuclear lysate of the human MCF7 breast epithelial adenocarcinoma cells. The cross-linking (X-ChiP) technique was used, crosslinking was performed for 15 minutes in formaldehyde. The primary antibody was used in concentration of 0.2 µg/µg chromatin and incubated with the sample for 16 hours at 4°C in SDS, DOC, TritonX-100, EDTA, HEPES, NaCl. The immunoprecipitated DNA was quantified by real time PCR. Ct values were converted to DNA copy numbers using a standard curve in the Q-PCR step. The number of binding events detected for each test reaction was then calculated by taking into account the DNA copy number, cell equivalents of chromatin used in the ChIP and PCR, and primer pair amplification efficiency.

    See Abreview

  • IHC - Wholemount of Caenorhabditis elegans embryo labelling RNA polymerase II CTD repeat YSPTSPS with ab817. Sample was incubated with primary antibody (1/100 in PBS + 3% BSA + 0.1% Triton-X 100) for 24 hours at 4°C. ab150113, a goat anti-mouse Alexa Fluor® 488 (undiluted) was used as the secondary antibody.

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab817 (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 (ab817, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG, H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

Anti-RNA polymerase II CTD repeat YSPTSPS antibody [8WG16] - ChIP Grade (ab817)参考文献

This product has been referenced in:
  • Schertel C  et al. A large-scale, in vivo transcription factor screen defines bivalent chromatin as a key property of regulatory factors mediating Drosophila wing development. Genome Res 25:514-23 (2015). Drosophila melanogaster . Read more (PubMed: 25568052) »
  • Sun J  et al. Identification of in vivo DNA-binding mechanisms of Pax6 and reconstruction of Pax6-dependent gene regulatory networks during forebrain and lens development. Nucleic Acids Res 43:6827-46 (2015). Read more (PubMed: 26138486) »

See all 92 Publications for this product

Product Wall

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 500000 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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提交于 Oct 21 2016

Application Western blot
Sample African Green Monkey Cell lysate - whole cell (VERO E6)
Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
Specification VERO E6
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 Aug 18 2016

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)
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 Aug 18 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 guarantees this product to work in the species/application used in this Abreview.
Application Immunoprecipitation
Sample Mouse Cell lysate - whole cell (mouse embryonic fibroblasts (MEFs))
Total protein in input 2e+006 cells
Immuno-precipitation step Other - Protein G Dynabeads
Specification mouse embryonic fibroblasts (MEFs)
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提交于 May 13 2016

Application Western blot
Sample Mouse Cell lysate - whole cell (Mouse embryonic fibroblasts)
Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
Loading amount 15000 cells
Treatment With or without 100uM zVAD
Specification Mouse embryonic 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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Dr. Yi-Chieh Perng

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提交于 Dec 07 2015

Application Western blot
Sample Human 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 24 2015

Application ChIP
Detection step Real-time PCR
Sample Human cytomegalovirus Cell lysate - nuclear (Human cytomegalovirus infected HF cells)
Specification Human cytomegalovirus infected HF cells
Negative control mouse IgG used as control antibody (from the kit (Invitrogen Magnify Chromatin Immunoprecipitation System))
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 5 minute(s) and 0 second(s)
Specification of the cross-linking agent: 37.5% formaldehyde
Positive control Input sample
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Dr. Yi-Chieh Perng

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提交于 Mar 25 2015

Application Immunocytochemistry/ Immunofluorescence
Blocking step (agent) for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Sample Quail Cell (Fibroblast QT6 cells)
Specification Fibroblast QT6 cells
Permeabilization Yes - 0.1% Triton
Fixative Paraformaldehyde
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提交于 Jul 31 2014

Application IHC - Wholemount
Sample Caenorhabditis elegans Embryo (C. elegans embryo)
Specification C. elegans embryo
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提交于 Mar 18 2014

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