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14934
Phospho-Rpb1 CTD (Thr4) Antibody
Primary Antibodies
Polyclonal Antibody

Phospho-Rpb1 CTD (Thr4) Antibody #14934

Citations (1)
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  1. WB
Western blot analysis of extracts from various cell lines using Phospho-Rpb1 CTD (Thr4) Antibody.
Peptide dot blot analysis demonstrating Phospho-Rpb1 CTD (Thr4) Antibody specificity. Antibody binding to pre-coated Rpb1 CTD peptides is shown using Phospho-Rpb1 CTD (Ser7) (E2B6W) Rabbit mAb #13780, Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb #13499, Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb #13523, and Phospho-Rpb1 CTD Thr4 Antibody #14934. As expected, the Phospho-Rpb1 CTD (Thr4) Antibody only binds to phospho-Rpb1 CTD peptide when phosphorylated at Thr4.
To Purchase # 14934
Cat. # Size Qty. Price
14934S
100 µl

Supporting Data

REACTIVITY H M R Mk
SENSITIVITY Endogenous
MW (kDa) 250
SOURCE Rabbit

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.

Protocol

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 10

Specificity / Sensitivity

Phospho-Rpb1 (Thr4) Antibody recognizes endogenous levels of Rpb1 protein only when the carboxy-terminal domain (CTD) heptapeptide repeat [Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, Ser7] is phosphorylated at Thr4. This antibody does not cross-react with Rpb1 CTD phosphorylated at Ser2, Ser5 or Ser7.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Polyclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr4 of human Rpb1 CTD heptapeptide repeat. Antibodies are purified by protein A and peptide affinity chromatography.

Background

RNA polymerase II (RNAPII) is a large multi-protein complex that functions as a DNA-dependent RNA polymerase, catalyzing the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates (1). The largest subunit, RNAPII subunit B1 (Rpb1), also known as RNAPII 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-terminal domain (CTD) of the protein (1). This CTD heptapeptide repeat is subject to multiple post-translational modifications, which dictate the functional state of the polymerase complex. Phosphorylation of the CTD during the active transcription cycle integrates transcription with chromatin remodeling and nascent RNA processing by regulating the recruitment of chromatin modifying enzymes and RNA processing proteins to the transcribed gene (1). During transcription initiation, RNAPII contains a hypophosphorylated CTD and is recruited to gene promoters through interactions with DNA-bound transcription factors and the Mediator complex (1). The escape of RNAPII from gene promoters requires phosphorylation at Ser5 by CDK7, the catalytic subunit of transcription factor IIH (TFIIH) (2). Phosphorylation at Ser5 mediates the recruitment of RNA capping enzymes, in addition to histone H3 Lys4 methyltransferases, which function to regulate transcription initiation and chromatin structure (3,4). After promoter escape, RNAPII proceeds down the gene to an intrinsic pause site, where it is halted by the negative elongation factors NELF and DSIF (5). At this point, RNAPII 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 (6). Phosphorylation at Ser2 creates a stable transcription elongation complex and facilitates recruitment of RNA splicing and polyadenylation factors, in addition to histone H3 Lys36 methyltransferases, which function to promote elongation-compatible chromatin (7,8). Ser2/Ser5-phosphorylated RNAPII then transcribes the entire length of the gene to the 3' end, where transcription is terminated. RNAPII dissociates from the DNA and is recycled to the hypophosphorylated form by various CTD phosphatases (1).
In addition to Ser2/Ser5 phosphorylation, Ser7 of the CTD heptapeptide repeat is also phosphorylated during the active transcription cycle. Phosphorylation at Ser7 is required for efficient transcription of small nuclear (sn) RNA genes (9,10). snRNA genes, which are neither spliced nor poly-adenylated, are structurally different from protein-coding genes. Instead of a poly(A) signal found in protein-coding RNAs, snRNAs contain a conserved 3'-box RNA processing element, which is recognized by the Integrator snRNA 3' end processing complex (11,12). Phosphorylation at Ser7 by CDK7 during the early stages of transcription facilitates recruitment of RPAP2, which dephosphorylates Ser5, creating a dual Ser2/Ser7 phosphorylation mark that facilitates recruitment of the Integrator complex and efficient processing of nascent snRNA transcripts (13-15).
Phosphorylation of the Rpb1 CTD heptapeptide repeat at Thr4 in is highly conserved from yeast to mammals. However, research studies using Thr4 phosphorylation-mutant Rpb1 proteins suggest different roles for this modification among species. While phosphorylation of Thr4 in yeast is not essential (16,17), Thr4 mutants in chicken and mammalian systems result in RNA processing errors and global defects in RNA elongation (18,19). Threonine 4 is directly phosphorylated by polo-kinase3 (PLK3) and cyclin dependent kinase-9 (CDK9) activity is thought to either directly or indirectly lead to the phosphorylation of this site (18,19).

  1. Brookes, E. and Pombo, A. (2009) EMBO Rep 10, 1213-9.
  2. Komarnitsky, P. et al. (2000) Genes Dev 14, 2452-60.
  3. Ho, C.K. and Shuman, S. (1999) Mol Cell 3, 405-11.
  4. Ng, H.H. et al. (2003) Mol Cell 11, 709-19.
  5. Cheng, B. and Price, D.H. (2007) J Biol Chem 282, 21901-12.
  6. Marshall, N.F. et al. (1996) J Biol Chem 271, 27176-83.
  7. Krogan, N.J. et al. (2003) Mol Cell Biol 23, 4207-18.
  8. Proudfoot, N.J. et al. (2002) Cell 108, 501-12.
  9. Chapman, R.D. et al. (2007) Science 318, 1780-2.
  10. Egloff, S. et al. (2007) Science 318, 1777-9.
  11. Egloff, S. et al. (2008) Biochem Soc Trans 36, 590-4.
  12. Baillat, D. et al. (2005) Cell 123, 265-76.
  13. Akhtar, M.S. et al. (2009) Mol Cell 34, 387-93.
  14. Egloff, S. et al. (2010) J Biol Chem 285, 20564-9.
  15. Egloff, S. et al. (2012) Mol Cell 45, 111-22.
  16. Stiller, J.W. et al. (2000) Yeast 16, 57-64.
  17. Schwer, B. and Shuman, S. (2011) Mol Cell 43, 311-8.
  18. Hsin, J.P. et al. (2011) Science 334, 683-6.
  19. Hintermair, C. et al. (2012) EMBO J 31, 2784-97.

Pathways

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