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POLR2A antibody

Principal name

POLR2A antibody

Alternative names for POLR2A antibody

RNA polymerase II, RNA polymerase II subunit B1, POLR2, POLRA, RPOL2, RPB1, RPB220, DNA-directed RNA polymerase II subunit RPB1, RNA-directed RNA polymerase II subunit RPB1, DNA-directed RNA polymerase II largest subunit, RNA polymerase II 220 kd

SwissProt ID

P04050 (Yeast), P04052 (Drome), P08775 (Mouse), P16356 (Caeel), P18616 (Arath), P24928 (Human), P36594 (Schpo)

Gene ID

5430 (POLR2A), 20020 (Polr2a)

Ncbi ID

NP_000928, 9606, NP_000928.1, 5874, NP_033115.1, NM_000937

Available reactivities

Dros (Drosophila), Hu (Human), Ms (Mouse), Rt (Rat), Hst (Hamster), Ye (Yeast), Can (Canine), Ze (Zebrafish), Chk (Chicken), Bov (Bovine), Eq (Equine), GP (Guinea Pig), Rb (Rabbit), S. cerevisiae (Saccharomyces cerevisiae)

Available hosts

Mouse, Rabbit

Available applications

Frozen Sections (C), Immunocytochemistry/Immunofluorescence (ICC/IF), Western blot / Immunoblot (WB), Enzyme Immunoassay (E), Paraffin Sections (P), ChIP assay (ChIP), Immunoprecipitation (IP), Flow Cytometry (F)

Background of POLR2A antibody

R polymerase II (RPII) is a large multi-protein complex that functions as a D-dependent R polymerase, catalyzing the transcription of D into R using the four ribonucleoside triphosphates as substrates. The largest subunit, RPII subunit B1 (Rpb1), also known as RPII subunit A (POLR2A), contains a unique heptapeptide sequence (Tyr1,Ser2,Pro3,Thr4,Ser5,Pro6,Ser7), which is repeated up to 52 times in the carboxy-termil domain (CTD) of the protein. This CTD heptapeptide repeat is subject to multiple post-translatiol modifications, which dictate the functiol state of the polymerase complex. Phosphorylation of the CTD during the active transcription cycle integrates transcription with chromatin remodeling and scent R processing by regulating the recruitment of chromatin modifying enzymes and R processing proteins to the transcribed gene. During transcription initiation, RPII contains a hypophosphorylated CTD and is recruited to gene promoters through interactions with D-bound transcription factors and the Mediator complex. The escape of RPII from gene promoters requires phosphorylation at Ser5 by CDK7, the catalytic subunit of transcription factor IIH (TFIIH). Phosphorylation at Ser5 mediates the recruitment of R capping enzymes, in addition to histone H3 Lys4 methyltransferases, which function to regulate transcription initiation and chromatin structure. After promoter escape, RPII proceeds down the gene to an intrinsic pause site, where it is halted by the negative elongation factors NELF and DSIF. At this point, RPII is unstable and frequently aborts transcription and dissociates from the gene. Productive transcription elongation requires phosphorylation at Ser2 by CDK9, the catalytic subunit of the positive transcription elongation factor P-TEFb. Phosphorylation at Ser2 creates a stable transcription elongation complex and facilitates recruitment of R splicing and polyadenylation factors, in addition to histone H3 Lys36 methyltransferases, which function to promote elongation-compatible chromatin.

Immunohistochemistry analysis of POLR2A antibody (Cat.-No. AP06407PU-N) in paraffin-embedded human breast carcinoma tissue.

AP06407PU-N

Western blot analysis of POLR2A antibody (Cat.-No. AP06407PU-N) in extracts from COS7 cells treated with EGF 200ng/ml 30'.

AP06407PU-N

ChIP results obtained with the POLR2A monoclonal antibody ( Cat # MAB0120 ) . ChIP assays were performed using MCF-7 cells ( Breast carcinoma cell line ) treated with estradiol ( MCF-7 + E2 ) or not treated ( MCF-7 ) .

MAB0120

Immunohistochemistry analysis of paraffin-embedded human breast carcinoma, using POLR2A (Phospho-Ser1619) Antibody. The picture on the right is treated with the synthesized peptide.

Immunofluorescence analysis of HeLa cells treated with PMA 125ng/ml 30', using POLR2A (Phospho-Ser1619) Antibody. The picture on the right is treated with the synthesized peptide.

Western blot analysis of extracts from COS7 cells treated with EGF 200ng/ml 30', using POLR2A (Phospho-Ser1619) Antibody. The lane on the right is treated with the synthesized peptide.

Immunohistochemistry analysis of paraffin-embedded human breast carcinoma tissue, using POLR2A (Ab-1619) Antibody. The picture on the right is treated with the synthesized peptide.

Immunofluorescence analysis of HeLa cells, using POLR2A (Ab-1619) Antibody. The picture on the right is treated with the synthesized peptide.

Western blot analysis of extracts from COS7 cells, treated with EGF 200ng/ml 30', using POLR2A (Ab-1619) Antibody. The lane on the right is treated with the synthesized peptide.

Immunohistochemical analysis of Rpb1 CTD (pSer5) staining in human breast cancer formalin fixed paraffin embedded tissue section. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0). The section was then incubated with the antibody at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Immunofluorescent analysis of Rpb1 CTD (pSer5) staining in HeLa cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with the primary antibody in 3% BSA-PBS and incubated overnight at 4 C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight 594-conjugated secondary antibody (red) in PBS at room temperature in the dark. DAPI was used to stain the cell nuclei (blue).

Western blot analysis of Rpb1 CTD (pSer5) expression in HeLa (A); A431 (B); NIH3T3 (C); PC12 (D) whole cell lysates.

Western Blot: RNA polymerase II Antibody (4H8) - Western blot analysis on NIH/3T3 cell lysate using anti-RNA polymerase II.

TA309814

Western blot analysis of extracts from COS7 cells treated with EGF (200ng/ml, 30min), using POLR2A (Ab-1619) antibody (#TA326076, Line 1 and 2).The lane on the right is treated with the synthesized peptide.

TA326076

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using POLR2A (Ab-1619) antibody (#TA326076).The picture on the right is treated with the synthesized peptide.

TA326076

Immunofluorescence analysis of HeLa cells, treated with PMA (125ng/ml, 30mins), using POLR2A (Ab-1619) antibody (#TA326076).The picture on the right is treated with the synthesized peptide.

TA326076

Western blot analysis of extracts of HeLa cell lines, using POLR2A antibody.

TA327272

Immunofluorescence analysis of HepG2 cell using POLR2A antibody. Blue: DAPI for nuclear staining.

TA327272

Western Blot: RNA polymerase II [p Thr4] Antibody [TA336792] - Flag-tagged Rpb1 was IP'd with Flag antibody. Western blotting was performed using P-thr4 antibody. 26r: Rpb1 with 26 all-consensus YSPTSPS repeat CTD. T4V: Rpb1 with 30 YSPVSPS repeat CTD.

TA336792

Western Blot: RNA polymerase II [p Thr4] Antibody [TA336792] - WB analysis of RNA pol II CTD pT4 in A. DT40 cell lysate and B. HeLa cell lysate.

TA336792

Dot Blot: RNA polymerase II [p Thr4] Antibody [TA336792] - Dot blot analysis of RNA pol II CTD pT4 using phosphorylated and non-phosphorylated peptides.

TA336792

Host: Rabbit; Target Name: POLR2A; Sample Tissue: Hela whole cell lysates; Antibody Dilution: 1.0 ug/ml

TA343189

WB using the antibody against Pol II 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.

TA347267

ChIP assays using HeLa cells: using sheared chromatin from 1 million cells. A titration 1, 2, 5 and 10 ug of ab per ChIP experiment was analyzed. IgG (2 ug/IP) was negative control. Primers used were specific for the promoter and the coding region of the constitutively expressed GAPDH and ACTB as positive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeat as negative controls. Image shows the recovery, expressed as a % of input (the relative amount of IP'd DNA compared to input DNA after qPCR).

TA347267

ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 1 ug of the ab against Pol II. The IP'd DNA was subsequently analysed on an Illumina Genome Analyzer. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Image shows the peak distribution along the complete sequence and a 400 kb region of the X-chromosome (figure 2A and B, respectively), and in a two genomic regions surrounding the GAPDH and ACTB positive control genes (figure 2C and D).

TA347267

Cross reactivity of the antibody against Pol II To test the specificity an ELISA was performed using a serial dilution of the antibody against Pol II. The wells were coated with peptides containing the unmodified C-terminal repeat sequence as well as different phosphorylated peptides. Image shows that the antibody recognizes the unphosphorylated Pol II as well as most phosphorylated forms.

TA347267

HeLa cells were stained with the antibody against Pol II and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the Pol II antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

TA347267

WB using the antibody against Pol II S2p 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.

TA347268

ChIP assays using HeLa cells & Pol II S2p ab. ChIP-seq” kit , using sheared chromatin from 1 million cells. A titration of 1, 2, 5 and 10 ug of ab was used. IgG (2 ug/IP) was negative control. qPCR was with primers specific for the coding region of the constitutively expressed GAPDH and ACTB as positive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeat as negative controls. Image shows the recovery, expressed as a % of input (the relative amount of IP'd DNA compared to input DNA after qPCR).

TA347268

ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 1 ug of the ab against Pol II S2p. The IP'd DNA was subsequently analysed on an Illumina Genome Analyzer. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Image shows the peak distribution along the complete sequence and a 150 kb region of the X-chromosome (figure 2A and B, respectively), and in a two genomic regions surrounding the GAPDH and ACTB positive control genes (figure 2C and D).

TA347268

Cross reactivity of the antibody against Pol IIS2p To test the specificity an ELISA was performed using a serial dilution of the antibody against Pol IIS2p. The wells were coated with peptides containing the unmodified C-terminal repeat sequence as well as different phosphorylated peptides. Image shows the specificity of the antibody for the S2 phosphorylation.

TA347268

HeLa cells were stained with the antibody against Pol II S2p and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the Pol II S2p antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

TA347268

WB using the antibody against Pol II S5p 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.

TA347269

ChIP assays using HeLa cells: ChIP-seq” kit, using sheared chromatin from 1 million cells. A titration of 1, 2, 5 and 10 ug ab was used. IgG (2ug/IP) was negative control. qPCR primers were specific for the promoter and the coding region of the constitutively expressed GAPDH and ACTB genesas positive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeatas negative controls. Image shows the recovery, expressed as a % of input (the relative amount of IP'd DNA compared to input DNA after qPCR).

TA347269

ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 1 ug of the ab against Pol II S5p. The IP'd DNA was subsequently analysed on an Illumina Genome Analyzer. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Image shows the peak distribution along the complete sequence and a 150 kb region of the X-chromosome (figure 2A and B, respectively), and in a two genomic regions surrounding the GAPDH and ACTB positive control genes (figure 2C and D).

TA347269

Cross reactivity of the antibody against Pol IIS5p To test the specificity an ELISA was performed using a serial dilution of the antibody against Pol IIS5p . The wells were coated with peptides containing the unmodified C-terminal repeat sequence as well as different phosphorylated peptides. Image shows the specificity of the antibody for the S5 phosphorylation.

TA347269

HeLa cells were stained with the antibody against Pol II S5p and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the Pol II S5p antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

TA347269

ICC staining of H14 on HeLa cells using 4% paraformaldehyde fixation.

TA352124

Primary Antibodies

Catalog No.	Host	Iso.	Clone	Pres.	React.	Applications
TA309814	POLR2A antibody
	Mouse Monoclonal RNA polymerase II Antibody (4H8)
	Mouse	IgG1	4H8	Purified	Hu, Ms, Ye	E, ICC/IF, IP, WB	0.1 ml / €415.00
							OriGene Technologies, Inc.
TA326076	POLR2A antibody
	Rabbit polyclonal POLR2A (Ab-1619) antibody
	Rabbit	IgG		Purified	Hu, Ms, Rt	WB	0.1 ml / €335.00
							OriGene Technologies, Inc.
+2 additional images
TA327272	POLR2A antibody
	POLR2A Rabbit Polyclonal Antibody
	Rabbit	IgG		Purified	Hu, Ms, Rt		0.1 mg / €335.00
							OriGene Technologies, Inc.
+1 additional image
TA336792	POLR2A antibody
	Rabbit Polyclonal RNA Polymerase II/POLR2A [p Thr4] Antibody
	Rabbit			Purified	Chk, Dros, Hu, Ye	WB	0.1 ml / €485.00
							OriGene Technologies, Inc.
+2 additional images
TA347267	POLR2A antibody
	Mouse Monoclonal Pol II Antibody
	Mouse	IgG1	Monoclol	Purified	Hu	E	50 µg / €395.00
							OriGene Technologies, Inc.
+4 additional images
TA347268	POLR2A antibody
	Mouse Monoclonal Pol II S2p Antibody
	Mouse	IgG1	Monoclol	Purified	Hu	E	50 µg / €395.00
							OriGene Technologies, Inc.
+4 additional images
TA347269	POLR2A antibody
	Mouse Monoclonal Pol II S5p Antibody
	Mouse	IgG1	Monoclol	Purified	Hu	E	50 µg / €395.00
							OriGene Technologies, Inc.
+4 additional images
AP06407PU-N	POLR2A antibody
	Rabbit			Aff - Purified	Hu, Ms, Rt	E, P, WB	0.1 mg / €370.00
							OriGene Technologies GmbH
+1 additional image

CRISPR

Catalog No.	Species	Donor Vector
KN216765	POLR2A - human gene knockout kit via CRISPR
	Human	GFP-Puro	1 kit / €1,290.00
			OriGene Technologies, Inc.
KN216765BN	POLR2A - human gene knockout kit via CRISPR
	Human	mBFP-Neo	1 kit / €1,290.00
			OriGene Technologies, Inc.
KN216765LP	POLR2A - human gene knockout kit via CRISPR
	Human	Luciferase-Puro	1 kit / €1,290.00
			OriGene Technologies, Inc.
KN216765RB	POLR2A - human gene knockout kit via CRISPR
	Human	RFP-BSD	1 kit / €1,290.00
			OriGene Technologies, Inc.

Human Lenti ORF Particles

Catalog No.
RC216765L1V	Lenti ORF particles, POLR2A (Myc-DDK tagged) - Human polymerase (RNA) II (DNA directed) polypeptide A, 220kDa (POLR2A), 200 uL, >10^7 TU/mL
		200 µl / €2,420.00
		OriGene Technologies, Inc.

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