Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

Histone H2A (D6O3A) Rabbit mAb #12349

Applications Reactivity Sensitivity MW (kDa) Isotype
W IF-IC ChIP H M R Mk (Hm) (B) (Dg) (GP) Endogenous 14 Rabbit IgG

Applications Key:  W=Western Blotting  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  B=Bovine  Dg=Dog  GP=Guinea Pig
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Histone H2A (D6O3A) Rabbit mAb recognizes endogenous levels of total histone H2A protein. This antibody does not cross-react with other histone proteins.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the carboxy terminus of human histone H2A protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Histone H2A (D6O3A) Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells using Histone H2A (D6O3A) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 5 μl of Histone H2A (D6O3A) 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 GAPDH Exon 1 Primers #5516, SimpleChIP® Human RPL30 Exon 3 Primers #7014, SimpleChIP® Human MYT-1 Exon 1 Primers #4493, 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, which is equivalent to one.


Background

Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4), is the primary building block of chromatin (1). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (2-5). These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and, therefore, gene expression (6). In most species, histone H2B is primarily acetylated at Lys5, 12, 15, and 20 (4,7). Histone H3 is primarily acetylated at Lys9, 14, 18, 23, 27, and 56. Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms (2,3). Phosphorylation at Ser10, Ser28, and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis (8-10). Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin. Immunostaining with phospho-specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase (11).

  1. Workman, J.L. and Kingston, R.E. (1998) Annu Rev Biochem 67, 545-79.
  2. Hansen, J.C. et al. (1998) Biochemistry 37, 17637-41.
  3. Strahl, B.D. and Allis, C.D. (2000) Nature 403, 41-5.
  4. Cheung, P. et al. (2000) Cell 103, 263-71.
  5. Bernstein, B.E. and Schreiber, S.L. (2002) Chem Biol 9, 1167-73.
  6. Jaskelioff, M. and Peterson, C.L. (2003) Nat Cell Biol 5, 395-9.
  7. Thorne, A.W. et al. (1990) Eur J Biochem 193, 701-13.
  8. Hendzel, M.J. et al. (1997) Chromosoma 106, 348-60.
  9. Goto, H. et al. (1999) J Biol Chem 274, 25543-9.
  10. Preuss, U. et al. (2003) Nucleic Acids Res 31, 878-85.
  11. Dai, J. et al. (2005) Genes Dev 19, 472-88.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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