Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-Histone H3 (Ser10) Chemiluminescent Sandwich ELISA Kit #7303

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Kit Includes Volume Solution Color
Histone H3 Rabbit mAb coated microwells 96 tests
Biotinylated P-Histone H3 (S10) Detection Ab 5.5 ml Green
HRP-Linked Streptavidin 5.5 ml Red
Luminol/Enhancer Solution 3 ml Colorless
Stable Peroxide Buffer 3 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

The PathScan® Phospho-Histone H3 (Ser10) Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-histone H3 (Ser10) protein with a chemiluminescent readout. Chemiluminescent ELISAs often have a wider dynamic range and higher sensitivity than conventional chromogenic detection. This chemiluminescent ELISA, which is offered in low volume microplates, shows increased signal and sensitivity while using smaller samples. A Histone H3 Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, both nonphospho- and phospho-histone H3 proteins are captured by the coated antibody. Following extensive washing, Biotinylated Phospho-Histone H3 (Ser10) Detection mAb is added to detect the captured phospho-histone H3 (Ser10) protein. HRP-linked Streptavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of phospho-histone H3 (Ser10) protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

The PathScan® Phospho-Histone H3 (Ser10) Chemiluminescent Sandwich ELISA Kit detects endogenous levels of phospho-histone H3 (Ser10). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Sensitivity

Sensitivity

The relationship between protein concentration of lysates from Jurkat cells, untreated or treated with calyculin A (100 nM) and pervanadate (1 mM), and immediate light generation with chemiluminescent substrate is shown. Cells were lysed using Cell Lysis Buffer (10X) #9803. Graph inset corresponding to the shaded area shows high sensitivity and a linear response at the low protein concentration range.

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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