Mouse Monoclonal RNA polymerase II RPB1 phospho S5 antibody. Suitable for ChIP, Flow Cyt (Intra), ELISA, Dot, WB, ICC/IF and reacts with Human, Mouse, Rat, Synthetic peptide - Human samples. Cited in 362 publications.
IgG1
Mouse
pH: 7.5
Preservative: 0.02% Sodium azide
Constituents: PBS
Liquid
Monoclonal
ChIP | Flow Cyt (Intra) | ELISA | Dot | WB | ICC/IF | |
---|---|---|---|---|---|---|
Human | Tested | Tested | Expected | Expected | Tested | Tested |
Mouse | Expected | Expected | Expected | Expected | Tested | Expected |
Rat | Expected | Expected | Expected | Expected | Tested | Expected |
Arabidopsis thaliana | Predicted | Predicted | Predicted | Predicted | Predicted | Predicted |
Synthetic peptide - Human | Not recommended | Not recommended | Tested | Tested | Not recommended | Not recommended |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1-4 µg for 106 Cells | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Arabidopsis thaliana | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Synthetic peptide - Human | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1 µg for 106 Cells | Notes ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody. |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Arabidopsis thaliana | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Synthetic peptide - Human | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Synthetic peptide - Human | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human, Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Arabidopsis thaliana | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Synthetic peptide - Human | Dilution info 1/1000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human, Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Arabidopsis thaliana | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/1000 | Notes - |
Species Human | Dilution info 1/1000 | Notes - |
Species Rat | Dilution info 1/1000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Arabidopsis thaliana | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Synthetic peptide - Human | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/1000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Arabidopsis thaliana | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Synthetic peptide - Human | Dilution info - | Notes - |
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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. Regulation of gene expression levels depends on the balance between methylation and acetylation levels of tha CTD-lysines (By similarity). Initiation or early elongation steps of transcription of growth-factors-induced immediate early genes are regulated by the acetylation status of the CTD (PubMed:24207025). Methylation and dimethylation have a repressive effect on target genes expression (By similarity).(Microbial infection) 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.
DNA-directed RNA polymerase II subunit RPB1, RNA polymerase II subunit B1, DNA-directed RNA polymerase II subunit A, DNA-directed RNA polymerase III largest subunit, RNA-directed RNA polymerase II subunit RPB1, POLR2, POLR2A
Mouse Monoclonal RNA polymerase II RPB1 phospho S5 antibody. Suitable for ChIP, Flow Cyt (Intra), ELISA, Dot, WB, ICC/IF and reacts with Human, Mouse, Rat, Synthetic peptide - Human samples. Cited in 362 publications.
DNA-directed RNA polymerase II subunit RPB1, RNA polymerase II subunit B1, DNA-directed RNA polymerase II subunit A, DNA-directed RNA polymerase III largest subunit, RNA-directed RNA polymerase II subunit RPB1, POLR2, POLR2A
IgG1
Mouse
pH: 7.5
Preservative: 0.02% Sodium azide
Constituents: PBS
Liquid
Monoclonal
4H8
Affinity purification Protein G
Blue Ice
1-2 weeks
+4°C
-20°C
Upon delivery aliquot
Avoid freeze / thaw cycle
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
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Full details and terms and conditions can be found here:
Terms & Conditions.
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.
Blocking/Diluting Buffer and concentration
All lanes: Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution
Lane 1: MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 10 µg
Lane 2: MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate. Then the membrane was incubated with alkaline phosphatase at 10 µg
All lanes: HRP-conjugated goat anti-mouse IgG at 1/10000 dilution
Predicted band size: 217 kDa
Observed band size: 270 kDa
All lanes: Western blot - 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) at 10 µg
Lane 2: NIH 3T3 (Mouse) Whole Cell Lysate (ab52956) at 10 µg
Lane 3: PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) at 10 µg
Lane 4: MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate (ab28419) Then the membrane was incubated with alkaline phosphatase at 10 µg
Lane 5: NIH 3T3 (Mouse) Whole Cell Lysate (ab52956) Then the membrane was incubated with alkaline phosphatase at 10 µg
Lane 6: PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) Then the membrane was incubated with alkaline phosphatase at 10 µg
All lanes: HRP-conjugated goat anti-mouse IgG at 1/10000 dilution
Predicted band size: 217 kDa
Observed band size: 270 kDa
Exposure time: 8min
Blocking/Diluting Buffer and concentration 5% NFDM/TBST
All lanes: Western blot - 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 at 10 µg
Lane 2: NIH/3T3 (mouse embryonic fibroblast), whole cell lysate. Then the membrane was incubated with alkaline phosphatase at 10 µg
All lanes: HRP-conjugated goat anti-mouse IgG at 1/10000 dilution
Predicted band size: 217 kDa
Observed band size: 270 kDa
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.
All lanes: Western blot - 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 at 30 µg
Lane 2: HEK293T transfected with Tat containing plasmid whole cell lysate at 30 µg
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 kDa
Exposure time: 30s
ELISA using ab5408 at varying antibody concentrations.
Curve_SPL4 indicates binding to RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide ab18488).
Binding to the following peptides was much weaker: Curve_SPL5 RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide ab12793), Curve_SPL6 RNA polymerase II CTD repeat YSPTSPS peptide (ab12795).
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 .
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 22°C. The secondary antibody used was goat anti-mouse DyLight® 488 (IgG H+L) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control ab91353, 2μg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
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 Anti-beta Tubulin antibody - Loading Control ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) 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.
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