The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 1µg for 106 cells.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use 1-4µg for 106 cells.
Use at an assay dependent concentration. PubMed: 19703992
1/1000. Detects a band of approximately 260 kDa (predicted molecular weight: 217 kDa).
Use at an assay dependent concentration.
Use at an assay dependent concentration. See Abreviews.
Use 2-0.3 µg for µg of chromatin.
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.
Belongs to the RNA polymerase beta' chain family.
The C-terminal domain (CTD) serves as a platform for assembly of factors that regulate transcription initiation, elongation, termination and mRNA processing.
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.
Blocking/Diluting Buffer and concentration 5% NFDM/TBST
Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)This image is courtesy of Michael Mancini, Baylor College of Medicine
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 1/1000 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-mouse 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
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
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.
Chromatin was prepared from U2OS 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.
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).
Overlay histogram showing HeLa 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.
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 : HEK293T 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