Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) 抗体 [3E8] - ChIP Grade (ab252852)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rat monoclonal [3E8] to RNA polymerase II CTD repeat YSPTSPS (phospho S5) - ChIP Grade
- Suitable for: ICC/IF, WB, ChIP, Dot blot, Indirect ELISA
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
製品の概要
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製品名
Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [3E8] - ChIP Grade
RNA polymerase II CTD repeat YSPTSPS 一次抗体 製品一覧 -
製品の詳細
Rat monoclonal [3E8] to RNA polymerase II CTD repeat YSPTSPS (phospho S5) - ChIP Grade -
由来種
Rat -
アプリケーション
適用あり: ICC/IF, WB, ChIP, Dot blot, Indirect ELISAmore details
適用なし: IP -
種交差性
交差種: Mouse, Rat, Human -
免疫原
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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ポジティブ・コントロール
- WB: HeLa, RAW 264.7 and PC-12 whole cell lysates. ICC/IF: HeLa and RAW264.7 cells. ChIP: Chromatin prepared from HeLa cells.
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特記事項
This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
製品の特性
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製品の状態
Liquid -
保存方法
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
バッファー
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 0.05% BSA, 40% Glycerol (glycerin, glycerine) -
Concentration information loading...
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精製度
Ion Exchange Chromatography -
ポリ/モノ
モノクローナル -
クローン名
3E8 -
アイソタイプ
IgG2a -
研究分野
関連製品
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Related Products
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab252852の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
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ICC/IF |
Use a concentration of 1.29 µg/ml.
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WB |
Use a concentration of 0.645 µg/ml. Detects a band of approximately 260 kDa (predicted molecular weight: 217 kDa).
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ChIP |
Use 5 µg for 25 µg of chromatin.
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Dot blot |
Use a concentration of 0.645 µg/ml.
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Indirect ELISA |
Use at an assay dependent concentration.
Use at 250 ng/ml. |
特記事項 |
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ICC/IF
Use a concentration of 1.29 µg/ml. |
WB
Use a concentration of 0.645 µg/ml. Detects a band of approximately 260 kDa (predicted molecular weight: 217 kDa). |
ChIP
Use 5 µg for 25 µg of chromatin. |
Dot blot
Use a concentration of 0.645 µg/ml. |
Indirect ELISA
Use at an assay dependent concentration. Use at 250 ng/ml. |
ターゲット情報
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機能
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. -
配列類似性
Belongs to the RNA polymerase beta' chain family. -
ドメイン
The C-terminal domain (CTD) serves as a platform for assembly of factors that regulate transcription initiation, elongation, termination and mRNA processing. -
翻訳後修飾
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. -
細胞内局在
Nucleus. - Information by UniProt
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参照データベース
- Entrez Gene: 5430 Human
- Entrez Gene: 20020 Mouse
- Entrez Gene: 363633 Rat
- Omim: 180660 Human
- SwissProt: P24928 Human
- SwissProt: P08775 Mouse
- Unigene: 270017 Human
- Unigene: 16533 Mouse
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別名
- 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
see all
画像
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Chromatin was prepared from HeLa cells according to the Abcam Dual-X-ChIP protocol*.
Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab252852 (red), or 5 µg of Rat IgG2a ab18450 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab252852 at 1/500 (1.29 μg/mL) dilution , followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2 dilution (Green). Confocal image showing nuclear staining in HeLa cell line, the signal decreased after phosphatase treatment at 37°C for 2h. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/500 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) at 1/500 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
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RNA polymerase II CTD repeat YSPTSPS (phospho S5) labeled with ab252852 at 1/1000 (0.645 μg/mL) dilution.
Goat Anti-Rat IgG (H+L), HRP) (ab205720) at 1/5000 dilution was used as secondary antibody.
Lane 1: RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide
Lane 2: RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide
Lane 3: RNA polymerase II CTD repeat YSPTSPS (phospho T4) peptide
Lane 4: RNA polymerase II CTD repeat YSPTSPS (phospho Y1) peptide
Lane 5: RNA polymerase II CTD repeat YSPTSPS (phospho S7) peptide
Lane 6: RNA polymerase II CTD repeat YSPTSPS non-phopho peptide
Blocking and dilution buffer: 5% NFDM/TBST.
Exposure time: 3 minutes.
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All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [3E8] - ChIP Grade (ab252852) at 0.645 µg/ml
Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate (Untreated membrane)
Lane 2 : HeLa whole cell lysate (Phosphatase treated membrane)
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rat IgG (H+L), HRP) (ab205720) at 1/5000 dilution
Predicted band size: 217 kDa
Observed band size: 260 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 22745433 and 23071310).
Exposure time: 3 seconds.
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ab252852 was used at 0 - 1000 ng/mL.
Antigens were used at 1000 ng/mL.
An Alkaline Phosphatase-conjugated Anti-Rat IgG (H+L) wasused as secondary antibody at 1/1000 dilution.
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All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [3E8] - ChIP Grade (ab252852) at 0.645 µg/ml
Lane 1 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 2 : PC-12 whole cell lysate (Phosphatase treated membrane)
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rat IgG (H+L), HRP) (ab205720) at 1/5000 dilution
Predicted band size: 217 kDa
Observed band size: 260 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 22745433 and 23071310).
Exposure time: 3 seconds.
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW264.7 cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab252852 at 1/500 (1.29 μg/mL) dilution, followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in RAW264.7 cell line, the signal decreased after phosphatase treatment at 37°C for 2h. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/500 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) secondary antibody at 1/500 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.
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All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [3E8] - ChIP Grade (ab252852) at 0.645 µg/ml
Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate (Untreated membrane)
Lane 2 : RAW264.7 whole cell lysate (Phosphatase treated membrane)
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rat IgG (H+L), HRP) (ab205720) at 1/5000 dilution
Predicted band size: 217 kDa
Observed band size: 260 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 22745433 and 23071310).
Exposure time: 37 seconds.
プロトコール
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
データシートおよび資料
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SDS download
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Datasheet download
参考文献 (3)
ab252852 は 3 報の論文で使用されています。
- Wu X et al. Down-Regulation of CXXC5 De-Represses MYCL1 to Promote Hepatic Stellate Cell Activation. Front Cell Dev Biol 9:680344 (2021). PubMed: 34621736
- Voss K et al. Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II. Transcription 6:91-101 (2015). PubMed: 26566685
- Chapman RD et al. Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7. Science 318:1780-2 (2007). PubMed: 18079404