Cell Signaling Technology

Product Pathways - Chromatin Regulation

Acetyl-Histone H3 (Lys9/Lys14) Antibody #9677

Applications Reactivity Sensitivity MW (kDa) Source
W IP IHC-P IF-IC ChIP H M R Mk (All) Endogenous 17 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  ChIP=Chromatin IP
Reactivity Key:  H=Human  M=Mouse  R=Rat  All=All  Mk=Monkey
Species cross-reactivity is determined by Western blot.

Specificity / Sensitivity

Acetyl-Histone H3 (Lys9/Lys14) Antibody detects endogenous levels of histone H3 only when acetylated at lysine 9 or lysine 14 . This antibody does not cross-react with other acetylated histones.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with a synthetic peptide (KLH-coupled) corresponding to the amino terminus of histone H3 acetylated on lysines 9 and 14. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of lysates from HeLa and NIH/3T3 cells, treated with or without 400 nM TSA for 18 hours, using Acetyl-Histone H3 (Lys9/Lys14) Antibody.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Acetyl-Histone H3 (Lys9/Lys14) Antibody.

IF-IC

IF-IC

Confocal immunofluorescent analysis of NIH/3T3 cells, treated overnight with Trichostatin A (TSA) #9950 (left) or untreated (right), labeled with Acetyl-Histone H3 (Lys9/Lys14) Antibody (green). Actin filaments have been labeled with Alexa Fluor™ 555 phalloidin (red).


Chromatin IP

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells and either 20 μl of Acetyl-Histone H3 (Lys9/Lys14) Antibody or 1 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 RPL30 and GAPDH genes, the inactive MYOD, and the heterochromatic Alpha 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 four core histone proteins (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 and 23. 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, 545-579.
  2. Hansen, J.C. et al. (1998) Biochemistry 37, 17637-17641.
  3. Strahl, B.D. and Allis, C.D. (2000) Nature 403, 41-45.
  4. Cheung, P. et al. (2000) Cell 103, 263-271.
  5. Bernstein, B.E. and Schreiber, S.L. (2002) Chem. Biol. 9, 1167-1173.
  6. Jaskelioff, M. and Peterson, C.L. (2003) Nat. Cell Biol. 5, 395-399.
  7. Thorne, A.W. et al. (1990) Eur. J. Biochem. 193, 701-713.
  8. Hendzel, M.J. et al. (1997) Chromosoma 106, 348-360.
  9. Goto, H. et al. (1999) J. Biol. Chem. 274, 25543-25549.
  10. Preuss, U. et al. (2003) Nucleic Acids Res. 31, 878-885.
  11. Dai, J. et al. (2005) Genes Dev. 19, 472-488.

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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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