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

Overview

  • Product name
    Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade
    See all RNA polymerase II CTD repeat YSPTSPS primary antibodies
  • Description
    Mouse monoclonal [4H8] to RNA polymerase II CTD repeat YSPTSPS (phospho S5) - ChIP Grade
  • Specificity
    ELISA and peptide blocking show that ab5408 preferentially binds phospho S5 RNA polymerase II CTD repeat YSPTSPS.
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, ChIP, ChIP/Chip, WB, ELISA, IP, CHIPseqmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, African green monkey
    Predicted to work with: Hamster
  • Immunogen

    Synthetic peptide corresponding to Human RNA polymerase II CTD repeat YSPTSPS (phospho S5). The sequence is repeated multiple times in the C-terminal domain of RNA polymerase II.
    Database link: P24928

  • Positive control
    • Flow Cytometry: HeLa cells (methanol fixed, Tween-20 permeabilized).
  • General notes

Properties

Applications

Our Abpromise guarantee covers the use of ab5408 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC/IF 1/1000.
ChIP Use 1-4µg for 106 cells.
ChIP/Chip Use at an assay dependent concentration. PubMed: 19703992
WB Use a concentration of 1 µg/ml. Detects a band of approximately 260 kDa (predicted molecular weight: 217 kDa).
ELISA Use at an assay dependent concentration.
IP Use at an assay dependent concentration. See Abreviews.
CHIPseq Use 2-0.3 µg for µg of chromatin.

Target

  • Function
    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.
  • Sequence similarities
    Belongs to the RNA polymerase beta' chain family.
  • Domain
    The C-terminal domain (CTD) serves as a platform for assembly of factors that regulate transcription initiation, elongation, termination and mRNA processing.
  • Post-translational
    modifications
    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.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • 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

Images

  • 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.

  • 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
  • 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).
  • 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
    HRP-conjugated goat anti-mouse IgG at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size : 217 kDa
    Observed band size : 260 kDa (why is the actual band size different from the predicted?)


    Exposure time : 30 seconds

    This image is courtesy of an anonymous Abreview.

    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

  • 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.



  • Predicted band size : 217 kDa

    Lane 1: buffer.

    Lane 2: extract from human 293T cells as positive control.

    Lanes 3 and 4: egg extracts made from wild type N2 worms and worms grown on RNAi food against dicer.

    Lanes 5-8: intact worms in mentioned larval stage and eggs boiled directly in SDS-PAGE buffer and loaded.

    This image was kindly supplied as part of the review submitted by Shveta Bagga.

     

Protocols

References

This product has been referenced in:
  • Blank MF  et al. SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription. Nucleic Acids Res 45:2675-2686 (2017). Human . Read more (PubMed: 28426094) »
  • Raciti GA  et al. Specific CpG hyper-methylation leads to Ankrd26 gene down-regulation in white adipose tissue of a mouse model of diet-induced obesity. Sci Rep 7:43526 (2017). ChIP ; Mouse . Read more (PubMed: 28266632) »

See all 201 Publications for this product

Customer reviews and Q&As

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (NIH3T3)
Gel Running Conditions
Reduced Denaturing (5)
Loading amount
15 µg
Specification
NIH3T3
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Aug 21 2017

Application
Western blot
Sample
Human Cell lysate - whole cell (Hek293T)
Gel Running Conditions
Reduced Denaturing (7.5)
Loading amount
30 µg
Specification
Hek293T
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Aug 18 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa cells)
Permeabilization
Yes - 0.3% Triton X 100
Specification
HeLa cells
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: RT°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Aug 15 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Human Cell lysate - whole cell (fibroblast)
Specification
fibroblast
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Username

Abcam user community

Verified customer

Submitted Feb 01 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Hek293T cells)
Gel Running Conditions
Reduced Denaturing (7.5% SDS gel)
Loading amount
30 µg
Treatment
Transfected with Tat containing plasmid
Specification
Hek293T cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jan 20 2016

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa cells)
Permeabilization
Yes - 1% Triton X-100
Specification
HeLa cells
Blocking step
BSA as blocking agent for 45 minute(s) · Concentration: 0.5% · Temperature: 25°C
Fixative
Paraformaldehyde
Username

Yupen Chung

Verified customer

Submitted Jan 18 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (mouse embryonic stem cells (E14 mES))
Permeabilization
Yes - 0,25 % Triton in 1xPBS
Specification
mouse embryonic stem cells (E14 mES)
Blocking step
BSA as blocking agent for 20 minute(s) · Concentration: 0.5% · Temperature: RT°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jan 05 2016

Application
Immunocytochemistry
Sample
Mouse Cell (Retina Tissue)
Specification
Retina Tissue
Blocking step
Serum as blocking agent for 20 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Feb 11 2015

Application
ChIP
Detection step
Real-time PCR
Sample
Human Cell lysate - whole cell (HeLa Cells)
Specification
HeLa Cells
Negative control
Uninduced Sample
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% Formaldehyde
Positive control
Induced Sample
Username

Abcam user community

Verified customer

Submitted Jul 21 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
Sample
African Green Monkey Cell (COS7)
Specification
COS7
Permeabilization
Yes - Triton X100 0.3%
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jul 09 2014

1-10 of 50 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up