Cell Signaling Technology

Product Pathways - Chromatin Regulation

Histone H3 Antibody (ChIP Formulated) #2650

Applications Reactivity Sensitivity MW (kDa) Source
ChIP H M (R) (Mk) (C) (B) (Dm) (X) (Z) Endogenous 17 Rabbit

Applications Key:  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  C=Chicken  B=Bovine  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish
Species cross-reactivity is determined by Western blot.

Protocols

Specificity / Sensitivity

Histone H3 Antibody (ChIP Formulated) detects endogenous levels of total histone H3 protein. This antibody does not cross-react with other histones.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy-terminal sequence of human histone H3. Antibodies are purified by protein A and peptide affinity chromatography.

Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 10 μl of Histone H3 Antibody (ChIP Formulated) #2650 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 primers specific for the transcriptionally active GAPDH and RPL30 genes, the inactive MYT1 gene, and the heterochromatic α Satellite repeat element. 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, on 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 of 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 of H3 Thr3 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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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.

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