Polymerase (RNA) II (DNA Directed) Polypeptide A, 220kDa (POLR2A) antibody

Antigen: Polymerase (RNA) II (DNA Directed) Polypeptide A, 220kDa (POLR2A)

Synonyms: RPB1, RPO2, POLR2, POLRA, RPBh1, RPOL2, RpIILS, hsRPB1, hRPB220, MGC75453, Rpb1, 220kDa, Rpo2-1, 19.m02399, POLR2A

Clonality: Monoclonal (4H8)

Host: Mouse

Reactivity: Human, Mouse (Murine), Yeast

Application: ELISA, Immunocytochemistry (ICC), Immunofluorescence (IF), Immunoprecipitation (IP), Western Blotting (WB)

Catalog no.: ABIN112080

Additional Information

Alternative name: POLR2A

Gene ID: 5430

UniProt: P24928

Immunogen: 10 repeats of synthetic peptide: YSPTSPS using chemically synthesized phosphor-Ser5.

Cross-Reactivity: Human, Mouse (Murine), Yeast

Isotype: IgG1

Clone: 4H8

Description: RNA polymerase II carboxy-terminal domain (CTD) interacts with a large multisubunitcomplex that contains TATA-binding protein (TBP) and is an integral part of thetranscription initiation complex. Phosphorylation of RNA polymerase II's largest subunitC-terminal domain (CTD) is a key event during mRNA metabolism. Numerous enzymes,including cell cycle-dependent kinases and TFIIF-dependent phosphatases target the CTD.

Characteristics: Synonyms: RNA polymerase II subunit B1, POLR2, POLRA, RPOL2, RPB1, RPB220, DNA-directed RNApolymerase II subunit RPB1, RNA-directed RNA polymerase II subunit RPB1, DNA-directedRNA polymerase II largest subunit, RNA polymerase II 220 kd

Application Details

Protocol: Immunofluorescence protocol-Formaldehyde fixationCollect cells from T. c. unit and remove media from petri dish using suction. Wash with 1x PBS and remove. Incubate cells in pre-warm (37°C) Para-Formaldehyde for 12 minutes at room temperatureon an orbital shaker. Remove PFA and incubate in 0. 5% Triton X-IOO in 1x PBS for 5 minutes at roomtemperature. Prepare blocking reagent, this is also the antibody diluent. Wash cells 2x with 1x PBS at room temperature, for 4 minutes/wash on an orbital shaker. Block with 1 % NCS and 1x PBS for 30 minutes at room temperature. Prepare primary antibodies (50μl/coverslip) and moist staining chambers. Wash cells 2x with lx PBS at room temperature and air dry briefly. Incubate with primary antibody for 1 hr at room temperature in the dark in stainingchambers. During this time prepare the secondary antibody. Wash cells 5x with 1x PBS (5 beaker changes/5 counts in each beaker)Incubate with secondary antibody for 1 hour at room temperature in the dark in stainingchambers. Wash cells 5x with 1x PBS. Mount in Dapi. Solutions (prepare fresh the same day of staining). 1x Phosphate buffered saline. Blocking reagent: 1% NCS in 1x PBS (use fresh l0x PBS). Fixation solution: 3. 5% Para formaldehyde. 1. 75g PFA in 20 ml d. H20 plus 5 drops 1M NaOH. Stir on a hot plate at 50-60°C untildissolved. Add 4 drops IN HCI and check pH indicator strip. PH should be 7. 4. Completevolume with d. H20 to 25ml and add 25ml 2xPBS. Check pH before adding to cover slips. Immunofluorescence protocol - Methanol/acetone fixationCollect cells from T. C. unit and remove media from petri dish using suction. Wash with 1x PBS and remove. Fix cells with cold methanol: acetone 1: 1 for 10 minutes on ice. Prepare blocking reagent, this is also the diluent for the antibodies. Remove fixative and wash cells 3x with Ix PBS at RT, for 4 minutes/wash on orbital shaker. Block with 1% NCS and Ix PBS for 30 minutes at RT. Prepare primary antibodies (50μl/coverslip) and moist staining chambers. Wash cells 2x with 1 x PBS at RT and air dry for approximately 7 minutes. Incubate with primary antibody for 1 hr at RT in the dark in staining chambers. During thistime prepare secondary antibody. Wash cells 5x with 1x PBS (5 beaker changes/5 counts in each beaker)Incubate with secondary antibody for 1 hr at R T in the dark in staining chambers. Wash cells 5x with 1x PBS. Mount in Dapi. Solutions (prepare fresh the same day of staining)1x Phosphate buffered saline. Blocking reagent: 1% NCS in 1x PBS (use fresh 10x PBS). Fixation solution: methanol: acetone 1: 1 ice cold.

Application Notes: Suitable for Western blot, Immunoprecipitation, ELISA and Immunofluorescence. Other applications not tested. Optimal dilutions are dependent on conditions and should be determined by the user.

Concentration: 0.8 mg/ml

Purification: Purified

Buffer: PBS pH 7.2 with 0.09% sodium azide as preservative.

Storage: Store the antibody at 2-8°C for one month or (in aliquots) at -20°C for longer. Avoid repeated freezing and thawing. Shelf life: One year from despatch.

Restrictions: For Research Use only

Publications

Patturajan, Conrad, Bregman et al.: "Yeast carboxyl-terminal domain kinase I positively and negatively regulates RNA polymerase II carboxyl-terminal domain phosphorylation." in: The Journal of biological chemistry, Vol. 274, Issue 39, pp. 27823-8, 1999 (PubMed).

Kristjuhan, Walker, Suka et al.: "Transcriptional inhibition of genes with severe histone h3 hypoacetylation in the coding region." in: Molecular cell, Vol. 10, Issue 4, pp. 925-33, 2002 (PubMed).

Kristjuhan, Wittschieben, Walker et al.: "Spreading of Sir3 protein in cells with severe histone H3 hypoacetylation." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 100, Issue 13, pp. 7551-6, 2003 (PubMed).