Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade

• 产品名称

  Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5)抗体[4H8] - ChIP Grade
  参阅全部 RNA polymerase II CTD repeat YSPTSPS 一抗

• 描述

  小鼠单克隆抗体[4H8] to RNA polymerase II CTD repeat YSPTSPS (phospho S5) - ChIP Grade

• 宿主

  Mouse

• 经测试应用

  适用于: Flow Cyt (Intra), ICC/IF, Dot blot, ChIP, WB, ELISAmore details

• 种属反应性

  与反应: Mouse, Rat, Human
  预测可用于: Arabidopsis thaliana

• 免疫原

  Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

• 阳性对照

• Flow Cyt: HeLa cells. WB: MCF7, HEK-293T and NIH/3T3 whole cell lysate. ICC/IF: HeLa and MCF7 cells. Dot Blot: RNA polymerase II CTD repeat YSPTSPS (phospho S5) phospho peptide. ChIP: U-2 OS cells.

• 常规说明

  ChIP protocols:
  ChIP protocol for cross-linking ChIP (X-ChIP)
  Native ChIP protocol
  Chromatin preparation from tissues for ChIP
  ChIP troubleshooting
  ChIP tips and tricks guide

  
  

  This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.

  
  

  The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

  If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

• 形式

  Liquid

• 存放说明

  Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.

• 存储溶液

  pH: 7.50
  Preservative: 0.02% Sodium azide
  Constituent: PBS
  
  Contains 0.4M Arginine

• 纯度

  Affinity purified

• 克隆

  单克隆

• 克隆编号

  4H8

• 骨髓瘤

  Sp2/0-Ag14

• 同种型

  IgG1

• 轻链类型

  kappa

The Abpromise guarantee

Abpromise™承诺保证使用ab5408于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。

数据库链接

• Entrez Gene: 5430 Human

• Entrez Gene: 20020 Mouse

• Entrez Gene: 363633 Rat

• Omim: 180660 Human

• SwissProt: P18616 Arabidopsis thaliana

• SwissProt: P24928 Human

• SwissProt: P08775 Mouse

• Unigene: 270017 Human

• Unigene: 16533 Mouse

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

• 

  Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  ab5408 staining RNA polymerase II CTD repeat YSPTSPS (phospho S5) in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab5408 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 4% paraformaldehyde (10 min).Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• 

  Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution
  
  Lane 1 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate (ab28419)
  Lane 2 : NIH 3T3 (Mouse) Whole Cell Lysate (ab52956)
  Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957)
  Lane 4 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate (ab28419) Then the membrane was incubated with alkaline phosphatase
  Lane 5 : NIH 3T3 (Mouse) Whole Cell Lysate (ab52956) Then the membrane was incubated with alkaline phosphatase
  Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) Then the membrane was incubated with alkaline phosphatase
  
  Lysates/proteins at 10 µg per lane.
  
  Secondary
  All lanes : HRP-conjugated goat anti-mouse IgG at 1/10000 dilution
  
  Predicted band size: 217 kDa
  Observed band size: 270 kDawhy is the actual band size different from the predicted?
  
  
  Exposure time: 8 minutes
  

• 

  Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution (purified)
  
  Lane 1 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate
  Lane 2 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate. Then the membrane was incubated with alkaline phosphatase
  
  Lysates/proteins at 10 µg per lane.
  
  Secondary
  All lanes : HRP-conjugated goat anti-mouse IgG at 1/10000 dilution
  
  Predicted band size: 217 kDa
  Observed band size: 270 kDawhy is the actual band size different from the predicted?
  
  
  
  

  Blocking/Diluting Buffer and concentration

• 

  ELISA - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  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).

• 

  ChIP - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  Chromatin was prepared from U-2 OS (human bone osteosarcoma epithelial cell line) 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.

• 

  Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution
  
  Lane 1 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
  Lane 2 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate. Then the membrane was incubated with alkaline phosphatase
  
  Lysates/proteins at 10 µg per lane.
  
  Secondary
  All lanes : HRP-conjugated goat anti-mouse IgG at 1/10000 dilution
  
  Predicted band size: 217 kDa
  Observed band size: 270 kDawhy is the actual band size different from the predicted?
  
  
  
  

  Blocking/Diluting Buffer and concentration 5% NFDM/TBST
  
  

• 

  Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)This image is courtesy of an anonymous Abreview.

  All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/800 dilution
  
  Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
  Lane 2 : HEK293T transfected with Tat containing plasmid whole cell lysate
  
  Lysates/proteins at 30 µg per lane.
  
  Secondary
  All lanes : HRP-conjugated goat anti-mouse IgG at 1/5000 dilution
  
  Performed under reducing conditions.
  
  Predicted band size: 217 kDa
  Observed band size: 260 kDawhy is the actual band size different from the predicted?
  
  
  Exposure time: 30 seconds
  
  
  

  7.5% SDS gel.

  Blocked with 5% BSA for 1 hour at 25°C.

  Incubated with the primary antibody for 3 hours at 25°C in 1X TBST buffer.

  See Abreview

• 

  Dot Blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  Dot blot analysis of RNA polymerase II CTD repeat YSPTSPS (phospho S5) phospho peptide (Lane 1), RNA polymerase II CTD repeat YSPTSPS (phospho 2) phospho peptide (Lane 2) and RNA polymerase II CTD repeat YSPTSPS non-phospho peptide (Lane 3) labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) phospho peptide with ab5408 at a dilution of 1/1000 dilution (1ug/ml). A HRP-conjugated goat anti-mouse IgG was used as the secondary antibody at a dilution of 1/10,000 dilution.

  Blocking buffer: 5% NFDM/TBST. Dilution buffer: 5% NFDM /TBST .

• 

  Flow Cytometry (Intracellular) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) 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 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 IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.