Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

Rpb1 CTD (4H8) Mouse mAb #2629

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP ChIP H M R Mk (Hm) (Dm) (Sc) Endogenous 250 Mouse IgG1

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  Dm=D. melanogaster  Sc=S. cerevisiae
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Rpb1 CTD (4H8) Antibody detects endogenous levels of total Rpb1 protein (both phosphorylated and unphosphorylated forms).

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide containing 10 heptapeptide repeats [Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, Ser7] in which Ser5 is phosphorylated.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Rpb1 CTD (4H8) Mouse mAb.

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 μl of Rpb1 CTD (4H8) Mouse mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human RPL30 Exon 3 Primers #7014, SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human MyoD1 Exon 1 Primers #4490, and SimpleChIP® Human MYT-1 Exon 1 Primers #4493. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Background

RNA polymerase II is a complex of 12 proteins that participates in transcription, mRNA processing, and transcription-coupled DNA repair (1,2). The largest subunit, Rpb1, contains a unique heptapeptide: Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, Ser7, which is repeated up to 52 times in the carboxy-terminal domain (CTD) of Rpb1 (3). This CTD is phosphorylated by cyclin-dependent kinases (CDKs), p44/42 MAPK, DNA-PKcs, and c-Abl (4). DNA damage caused by UV-light, hydrogen peroxide, or cisplatin results in ubiquitination and proteasomal degradation of Rpb1 (1,3). The kinase inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole prevents ubiquitination of Rpb1, suggesting that CTD phosphorylation is required for proteolysis (5).

  1. Svejstrup, J.Q. (2002) Nat Rev Mol Cell Biol 3, 21-9.
  2. Shilatifard, A. et al. (2003) Annu Rev Biochem 72, 693-715.
  3. Inukai, N. et al. (2004) J Biol Chem 279, 8190-5.
  4. Oelgeschläger, T. (2002) J Cell Physiol 190, 160-9.
  5. Luo, Z. et al. (2001) Mutat Res 486, 259-74.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

Products