Phospho-POLR2A (S5) Recombinant Rabbit Monoclonal Antibody [JM51-21]
Catalog# ET1703-87
Phospho-POLR2A (S5) Recombinant Rabbit Monoclonal Antibody [JM51-21]
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WB
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IP
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IF-Cell
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IF-Tissue
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IHC-P
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FC
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Human
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Mouse
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Rat
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unconjugated
Overview
Product Name
Phospho-POLR2A (S5) Recombinant Rabbit Monoclonal Antibody [JM51-21]
Antibody Type
Recombinant Rabbit monoclonal Antibody
Immunogen
Synthetic phospho-peptide corresponding to residues surrounding Ser5 of Human POLR2A aa 1590-1627 / 1970.
Species Reactivity
Human, Mouse, Rat
Validated Applications
WB, IP, IF-Cell, IF-Tissue, IHC-P, FC
Molecular Weight
Predicted band size: 217 kDa
Positive Control
HeLa cell lysate, A431 cell lysate, MCF7 cell lysate, NIH/3T3 cell lysate, PC-12 cell lysate, HeLa, human kidney tissue, mouse kidney tissue, rat kidney tissue.
Conjugation
unconjugated
Clone Number
JM51-21
RRID
Product Features
Form
Liquid
Concentration
Storage Instructions
Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.
Storage Buffer
1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Isotype
IgG
Purification Method
Protein A affinity purified.
Application Dilution
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WB
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1:2,000
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IF-Cell
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1:500
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IHC-P
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1:200-1:1000
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FC
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1:1000
Target
Function
DNA-directed RNA polymerase II subunit RPB1, also known as RPB1, is an enzyme that is encoded by the POLR2A gene in humans. This gene encodes the largest subunit of RNA polymerase II, the polymerase responsible for synthesizing messenger RNA in eukaryotes. The product of this gene 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, this subunit, in combination with several other polymerase subunits, forms the DNA-binding domain of the polymerase, a groove in which the DNA template is transcribed into RNA.
Background References
1. Hernando H et al. EZH2 Inhibition Blocks Multiple Myeloma Cell Growth through Upregulation of Epithelial Tumor Suppressor Genes. Mol Cancer Ther 15:287-98 (2016).
2. Bolt MJ et al. Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-a protein levels and activity. Oncogene N/A:N/A (2014).
Sequence Similarity
Belongs to the RNA polymerase beta' chain family.
Post-translational Modification
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. Regulates 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, dimethylated and trimethylated. Methylation occurs in the earliest transcription stages and precedes or is concomitant to 'Ser-5' and 'Ser-7' phosphorylation. Dimethylation and trimehtylation at 'Lys-7' of non-consensus heptapeptide repeats are exclusively associated with phosphorylated CTD.; Ubiquitinated by WWP2 leading to proteasomal degradation (By similarity). Following UV treatment, the elongating form of RNA polymerase II (RNA pol IIo) is ubiquitinated on 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.
Subcellular Location
Nucleus, Cytoplasm, Chromosome.
Synonyms
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
ExpandImages
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☑ Cell treatment (CT)
Western blot analysis of Phospho-POLR2A (S5) on different lysates with Rabbit anti-Phospho-POLR2A (S5) antibody (ET1703-87) at 1/2,000 dilution.
Lane 1: HeLa cell lysate
Lane 2: A431 cell lysate
Lane 3: MCF7 cell lysate
Lane 4: NIH/3T3 cell lysate
Lane 5: PC-12 cell lysate
Lane 6: HeLa cell lysate, the membrane treated with λpp for 1 hour
Lysates/proteins at 20 µg/Lane.
Predicted band size: 217 kDa
Observed band size: 250 kDa
Exposure time: 17 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1703-87) at 1/50,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
Immunocytochemistry analysis of HeLa cells labeling Phospho-POLR2A (S5) with Rabbit anti-Phospho-POLR2A (S5) antibody (ET1703-87) at 1/500 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-POLR2A (S5) antibody (ET1703-87) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-Phospho-POLR2A (S5) antibody (ET1703-87) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1703-87) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-Phospho-POLR2A (S5) antibody (ET1703-87) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1703-87) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-Phospho-POLR2A (S5) antibody (ET1703-87) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1703-87) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Flow cytometric analysis of Phospho-POLR2A (S5) was done on Hela cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1703-87, 1/1000) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
Citation
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Effect of CDK7 inhibitor on MYCN-amplified retinoblastoma
Journal: Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms
DOI:
IF: 4.7
Application: WB,IHC-P
Reactivity: Human
Publish date: 2023 Sept
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