Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121

Kit Includes Volume Solution Color
Acetyl-Histone H3 (Lys9) Rabbit Antibody Coated Microwells 96 tests
Histone H3 Rabbit Detection Antibody 11 milliliters Green
HRP-Linked Streptavidin 11 milliliters Red
TMB Substrate 11 milliliters Colorless
STOP Solution 11 milliliters Colorless
Sealing Tape 2 sheets
20X Wash Buffer 25 milliliters Colorless
Sample Diluent 25 milliliters Blue
Cell Lysis Buffer (10X) # 9803 15 milliliters 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 (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M Mk

Reactivity Key:  H=Human  M=Mouse  Mk=Monkey

Description

The PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of histone H3 when acetylated at Lys9. An Acetyl-Histone H3 (Lys9) Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, acetyl-histone H3 (Lys9) is captured by the coated antibody. Following extensive washing, biotinylated Histone H3 Rabbit Antibody* is added to detect the histone H3 protein. HRP-linked streptavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of histone H3 acetylated at Lys9.* Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121 detects endogenous levels of histone H3 when acetylated at Lys9. As shown in Figure 1 using the Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121, a high level of acetylation at Lys9 on histone H3 is detected in COS cells when treated with TSA. The level of total histone H3 (modified and unmodified) remains unchanged as shown by Western analysis (Figure 1). Similar results are obtained when NIH/3T3 and Jurkat cells are treated with TSA (data not shown).

Sandwich ELISA

Sandwich ELISA

Figure 1. Treatment of COS cells with trichostatin A (TSA) increases the acetylation of Histone H3 at Lys9 detected by PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121. TSA treatment does not affect the level of histone H3 that is detected by Western analysis. COS cells (70-80% confluent) were treated for 16-18 hours with 0.4 μM TSA at 37°C. Absorbance readings at 450 nm are shown in the top figure while the corresponding Western blots using Histone H3 Antibody #9715 (left panel) or Acetyl-Histone H3 (Lys9) Antibody #9671 (right panel) are shown in the bottom figure.

Sandwich ELISA

Sandwich ELISA

Figure 2. The relationship between the protein concentration of the lysate from untreated and TSA-treated COS cells and the absorbance at 450 nm is shown.

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 (2,3). 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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