Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - Chromatin Regulation / Epigenetics

Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #3458

Applications Reactivity Isotype
IF-IC F H M R Mk Rabbit IgG

Applications Key:  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
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

Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) detects endogenous levels of histone H3 only when phosphorylated at Ser10. The antibody does not cross-react with other phosphorylated histones or with acetylated histones.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser10 of human histone H3. This antibody was conjugated to Alexa Fluor® 647 under optimal conditions with an F/P ratio of 2-6. The Alexa Fluor® 647 dye is maximally excited by red light (e.g. 633 nm He-Ne laser). Antibody conjugates of the Alexa Fluor® 647 dye produce bright far-red-fluorescence emission, with a peak at 665 nm.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (A) or taxol-treated (B), stained with Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate). The blue inserts represent PI (DNA) staining alone, showing an increase in the number of mitotic cells in the taxol-treated sample.

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells using Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) (blue pseudocolor), β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red) and Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (D13A11) XP® Rabbit mAb #2914 (green).

Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometry and immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb #3377.

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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Companion Products

The Alexa Fluor® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc., for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents.Alexa Fluor® is a registered trademark of Molecular Probes, Inc.

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

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