Cell Signaling Technology

Product Pathways - Chromatin Regulation / Epigenetics

Phospho-Histone H3 (Mitotic Marker) Antibody Sampler Kit #9849

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377 40 µl W IF-IC F H M R Mk Z 17 Rabbit IgG
Phospho-Histone H3 (Thr11) Antibody #9764 40 µl W IP IF-IC F H M R (X) 17 Rabbit
Phospho-Histone H3 (Ser28) Antibody #9713 40 µl W IP IF-F IF-IC F H M Hm Dm (R) (C) (X) (Z) (B) 17 Rabbit
Phospho-Histone H3 (Thr3) Antibody #9714 40 µl W IHC-P H M R 17 Rabbit
Histone H3 (D1H2) XP® Rabbit mAb #4499 40 µl W IHC-P IF-IC H M R Mk (Hm) (C) (Dm) (X) (Z) (B) 17 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-F=Immunofluorescence (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  C=Chicken  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

All antibodies in the Phospho-Histone H3 Antibody Sampler Kit recognize Histone H3 only when modified at the indicated site.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, either untreated or treated with nocodazole (0.1 mg/ml for 18 hours), using Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377 (upper) or Histone H3 Antibody #9715 (lower). Phospho-specificity of the antibody is shown by further treatment of the lysate with λ phosphatase.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Histone H3 (D1H2) XP® Rabbit mAb #4499.

Western Blotting

Western Blotting

Western blot analysis of extracts from CHO and HeLa cells, untreated or synchronized in metaphase by treatment with 0.1 mg/ml nocodazole for 4 h, followed by isolation of metaphase cells by mitotic shake-off. Blots were probed with Phospho-Histone H3 (Ser28) Antibody #9713 (upper) or Histone H3 Antibody #9715 (lower).


Western Blotting

Western Blotting

Western blot analysis of extracts from NIH/3T3 and HeLa cells, untreated or treated with calyculin A and 20% FCS, using Phospho-Histone H3 (Thr3) Antibody #9714 (upper) or Histone H3 Antibody #9715 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa, C6 and NIH/3T3 cells, untreated or treated with nocodazole for 24 hours in the presence or absence of λ phosphatase, using Phospho-Histone H3 (Thr11) Antibody #9764 (upper) or Histone H3 Antibody #9715 (lower).

Description

The Phospho-Histone H3 (Mitotic Marker) Antibody Sampler Kit provides a fast and economical means of evaluating phosphorylation sites associated with mitosis on Histone H3. The kit contains enough primary and secondary antibodies to perform four Western blots.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Thr3, Thr11 or Ser28 of human Histone H3. Antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3.

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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Protocols

* Product-specific protocol.

Companion Products

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

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