RNA pol II CTD Ser2ph / Ser5ph antibody (mAb)

Clone: 1A12G10
Aliases: RPB1, RPO2, POLR2, POLRA, RPBh1, RPOL2, RpIILS, hsRPB1, hRPB220
Catalog No: 61665 Format: 100 µg $445 Buy
Catalog No: 61666 Format: 10 µg $105 Buy

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Antibody Type:
Monoclonal
Isotype:
IgG1k
Purification:
Protein A Chromatography
Host:
Mouse
Molecular Weight:
240 kDa
Reactivity:
Human

Applications

ChIP Validated ChIP-Seq Validated Western Blot Validated Immunofluorescence Validated Immunocytochemistry Validated

Application Notes


Validated Applications:
ChIP: 5 - 10 µg per ChIP
ChIP-Seq: 5 - 10 µg each
ICC/IF: 5 µg/ml dilution
WB: 0.5 - 2 µl/ml dilution

ChIP-Seq validation was performed by Active Motif's Epigenetics Services and the complete data set is available in the UCSC Genome Browser by clicking here.

Note: many chromatin-bound proteins are not soluble in a low salt nuclear extract and fractionate to the pellet. Therefore, we recommend a High Salt / Sonication Protocol when preparing nuclear extracts for Western Blot.

Immunogen

This antibody was raised against a synthetic peptide containing phospho-serine 2 and phospho-serine 5 of the CTD of human RNA Polymerase II.

Buffer

Purified IgG in PBS with 30% glycerol and 0.035% sodium azide. Sodium azide is highly toxic.

References

 
RNA pol II CTD Ser2ph / Ser5ph antibody (mAb) tested by ChIP-Seq.

RNA pol II CTD Ser2ph / Ser5ph antibody (mAb) (Clone 1A12G10) tested by ChIP-Seq.
ChIP was performed using the ChIP-IT® High Sensitivity Kit (Cat. No. 53040) with 30 µg of chromatin from HL-60 cells and 4 µg of antibody. ChIP DNA was sequenced on the Illumina HiSeq and 11 million sequence tags were mapped to identify RNA pol II CTD Ser2ph / Ser5ph binding sites. The image shows binding across a region of chromosome 15. You can view the complete data set in the UCSC Genome Browser, starting at this specific location, here.

RNA pol II CTD Ser2ph / Ser5ph antibody (mAb) tested by immunofluorescence.

RNA pol II CTD Ser2ph / Ser5ph antibody (mAb) (Clone 1A12G10) tested by immunofluorescence.
Left: HeLa cell stained with RNA pol II CTD Ser2ph / Ser5ph antibody (mAb). Middle:Hoechst. Right: Merge.

RNA pol II CTD Ser2ph / Ser5ph antibody (mAb) tested by Western blot.

RNA pol II CTD Ser2ph / Ser5ph antibody (mAb) (Clone 1A12G10) tested by Western blot.
HeLa nuclear extract (30 µg per lane) probed with RNA pol II CTD Ser2ph / Ser5ph antibody (mAb) at a 1 µg/ml dilution.

Background

RNA pol II (RNA polymerase II) is responsible for synthesizing messenger RNA in eukaryotes. RNA pol II contains a carboxy terminal domain composed of heptapeptide repeats that are essential for polymerase activity. These repeats contain serine and threonine residues that are phosphorylated in actively transcribing RNA polymerase. In addition, RNA pol II, in combination with several other polymerase subunits, form the DNA binding domain of the polymerase, a groove in which the DNA template is transcribed into RNA.

During the transcription cycle, the CTD of the large subunit of RNA pol II is reversibly phosphorylated. RNA pol II containing unphosphorylated CTD is recruited to the promoter, whereas the hyperphosphorylated CTD form is involved in active transcription. Phosphorylation occurs at two sites within the heptapeptide repeat, at serine 2, serine 5 and serine 7. RNA pol II Serine 2 phosphorylation is enriched over the gene body and is associated with transcriptional elongation. RNA pol II Serine 5 phosphorylation is confined to promoter regions and is necessary for the initiation of transcription.

Chromatin IP Assays

This antibody has been validated for use in ChIP and/or ChIP-Seq, and can be used with Active Motif's ChIP-IT® High Sensitivity Kit or our magnetic bead-based ChIP-IT® Express Kits. For an overview of all of our ChIP products, please visit our Chromatin IP Page to learn about ChIP products designed for use with RNA pol II CTD Ser2ph / Ser5ph antibody (mAb).

Bridging Antibody to Improve ChIP, MeDIP and IP

Some isotypes of mouse IgG antibodies do not bind well to protein G-conjugated agarose/magnetic beads, which are commonly used in ChIP, MeDIP and IP experiments. Capture efficiency can be improved by including an anti-mouse IgG bridging antibody because it binds with a strong affinity to both the protein G beads and the mouse IgG antibody. By increasing the affinity of the mouse primary antibody for the protein G bead, the Bridging Antibody for Mouse IgG can help improve the results of all ChIP, MeDIP and IP experiments that use RNA pol II CTD Ser2ph / Ser5ph antibody (mAb).

Compatible Secondary Antibodies

Active Motif has developed a variety of high-quality secondary antibodies for most applications, with anti-rabbit and anti-mouse conjugates to a broad line of high-quality fluorescent molecules, as well as horse radish peroxidase (HRP). Visit our Secondary Antibody Conjugates page to find the perfect secondaries for immunofluorescence experiments with RNA pol II CTD Ser2ph / Ser5ph antibody (mAb).

Storage

Some products may be shipped at room temperature. This will not affect their stability or performance. Upon receipt, unconjugated antibodies may be stored at -20°C for up to 2 years. Fluorophore- & enzyme-conjugated antibodies should be stored at 4°C. Fluorophore-conjugated antibodies should be protected from light. Keep reagents on ice when not in storage; to avoid repeated freeze/thaw cycles, we recommend aliquoting items that will be stored frozen into single-use fractions prior to freezing.

Guarantee

This product is guaranteed for 6 months from date of receipt.

This product is for research use only and is not for use in diagnostic procedures.

Application Key

  • ChIP = Chromatin Immunoprecipitation;
  • DB = Dot Blot;
  • ELISA = Enzyme-linked Immunosorbent Assay;
  • EMSA = Electrophoretic Mobility Shift Assay
  • FC = Flow Cytometry;
  • ICC = Immunocytochemistry;
  • IF = Immunofluorescence;
  • IHC = Immunohistochemistry;
  • IP = Immunoprecipitation;
  • MeDIP = Methyl-DNA Immunoprecipitation;
  • TR-FRET = Time-Resolved Fluorescence Resonance Energy Transfer;
  • WB = Western Blotting