RNA polymerase II (pol II) is a key enzyme in the regulation and control of gene transcription. It is able to unwind the DNA double helix, synthesize RNA, and proofread the result. Pol II is a complex enzyme, consisting of 12 subunits, of which the B1 subunit (UniProt/Swiss-Prot entry P24928) is the largest. Together with the second largest subunit, B1 forms the catalytic core of the RNA polymerase II transcription machinery.
Pol II Antibody
Alternative names: POLR2A, RPB1, POLR2, RPOL2
Monoclonal antibody raised in mouse against the B1 subunit of RNA polymerase II (polymerase (RNA) II (DNA directed) polypeptide A) of wheat germ. Interacts with the highly conserved C-terminal domain of the protein containing the YSPTSPS repeat.
| Lot | 001 |
|---|---|
| Concentration | 2.2 µg/µl |
| Species reactivity | Human, Mouse, Xenopus: positive. Other species: not tested. |
| Type | Monoclonal. ChIP-grade. ChIP-seq grade. CUT&Tag-grade. |
| Purity | Protein A purified monoclonal antibody |
| Host | Mouse |
| Storage Conditions | Store at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles. |
| Storage Buffer | PBS containing 0.05 % Na-azide |
| Precautions | This product is for research use only. Not for use in diagnostic or therapeutic procedures. |
| Applications | Suggested dilution | References |
|---|---|---|
| ChIP/ChIP-seq* | 0.5 - 1 µl per ChIP | Fig 1, 2 |
| CUT&Tag | 1 µg | Fig 3 |
| Western Blotting | 1:1,000 | Fig 4 |
*Please note that of the optimal antibody amount per IP should be determined by the end-user. We recommend testing 0.5-5 µg per IP.
- Validation Data
Figure 1. ChIP results obtained with the monoclonal antibody directed against Pol II
ChIP assays were performed using human HeLa cells, the monoclonal antibody against Pol II (cat. No. C15200253) and optimized PCR primer pairs for qPCR. ChIP was performed with the iDeal ChIP-seq kit (cat. No. C01010051), using sheared chromatin from 1 million cells. A titration consisting of 0.5, 1, 2 and 5 µg of antibody per ChIP experiment was analyzed. IgG (2 µg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the GAPDH and EIF4A2 genes, used as positive controls, and for the MYOD1 gene and the Sat2 satellite repeat, used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
Figure 2. ChIP-seq results obtained with the monoclonal antibody directed against Pol II
ChIP was performed on sheared chromatin from 1 million HeLa cells using 0.5 µg of the antibody against Pol II (cat. No. C15200253) as described above. The IP’d DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the enrichment along the complete sequence and a 1.5 Mb region of chromosome 1 (fig 2A and B) and in genomic regions of chromosome 12 and 3, surrounding the GAPDH and EIF4A2 positive control genes (fig 2C and D).
Figure 3. Cut&Tag results obtained with the monoclonal antibody directed against Pol II
CUT&TAG was performed on 300,000 K562 cells using 1 µg of the monoclonal antibody against Pol II (cat. No. C15200253), the pA-Tn5 transposase (C01070001) and the Universal Cut&Tag kit (cat. No. C01070024). The libraries were subsequently analysed on an Illumina NovaSeq sequencer (2x50 paired-end reads) according to the manufacturer’s instructions. The tags were aligned to the human genome (hg38) using the BWA algorithm. Figure 3 shows the peak distribution in 2 genomic regions on the X-chromosome and on chromosome 1 (figure 3A and B, respectively).
Figure 4. Western blot analysis using the monoclonal antibody directed against Pol II
Whole cell extracts (40 µg) from HeLa cells transfected with Pol II siRNA (lane 2) and from an untransfected control (lane 1) were analysed by Western blot using the antibody against Pol II (cat. No. C15200253) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left. - 出版物
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