Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Chromatin Regulation / Epigenetics

Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb #8240

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP ChIP H M R Mk Endogenous 23 Rabbit IgG

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

Protocols

Specificity / Sensitivity

Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb recognizes endogenous levels of histone H2A protein only when ubiquitinated at Lys119. The antibody does not cross-react with other ubiquitinated proteins or free ubiquitin.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human histone H2A protein in which Lys119 is mono-ubiquitinated.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAb.

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 NCCIT cells and either 10 µl of Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit 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 HoxA1 Intron 1 Primers #7707, SimpleChIP® Human HoxA2 Promoter Primers #5517, SimpleChIP® Human GAPDH Exon 1 Primers #5516, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to one).

Background

The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (1). Ubiquitin is a conserved 76 amino acid peptide unit that can be covalently linked to many cellular proteins by the ubiquitination process. Three components are involved in this protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thioester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (2). Histone H2A is mono-ubiquitinated at Lys119 by the Polycomb Repressor Complex 1 (PRC1) and is critical for transcriptional silencing of the developmental HOX genes and X chromosome inactivation (3-6). PRC1 is composed of Bmi1 and RING1A (also RING1 or RNF1), both of which act to enhance the E3 ubiquitin ligase activity of the catalytic subunit RING1B (also RING2 or RNF2) (3,4). Histone H2A is also mono-ubiquitinated at Lys119 at sites of DNA damage. This mono-ubiquitination event requires the PRC1 components Bmi1 and RING1B, in addition to another E3 ubiquitin ligase RNF8, and contributes to subsequent recruitment of the BRCA1 complex, via binding of RAP80/UIMC1 (ubiquitin interactive motif containing 1 protein) (7-10).

  1. Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.
  2. Liu, F. and Walters, K.J. (2010) Trends Biochem Sci 35, 352-60.
  3. Wang, H. et al. (2004) Nature 431, 873-8.
  4. Cao, R. et al. (2005) Mol Cell 20, 845-54.
  5. de Napoles, M. et al. (2004) Dev Cell 7, 663-76.
  6. Fang, J. et al. (2004) J Biol Chem 279, 52812-5.
  7. Ginjala, V. et al. (2011) Mol Cell Biol 31, 1972-82.
  8. Bergink, S. et al. (2006) Genes Dev 20, 1343-52.
  9. Marteijn, J.A. et al. (2009) J Cell Biol 186, 835-47.
  10. Wu, J. et al. (2009) Mol Cell Biol 29, 849-60.

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