Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Pan-Methyl-Histone H3 (Lys9) Sandwich ELISA Kit #7864

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

Important Ordering Details: Product is assembled upon order to ensure maximum activity. Domestic: Please allow up to two weeks for your order to be processed and shipped. International: Please allow up to three weeks, depending on the country, for your order to be processed and shipped.

Kit Includes Volume Solution Color
Pan-Methyl-Histone H3(K9) Ab Coated Microwells 96 tests
Histone H3 Detection Ab 11 ml Green
HRP-linked Streptavidin 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
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 Mk

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

Description

The PathScan® Pan-Methyl-Histone H3 (Lys9) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of histone H3 when methylated at Lys9. A Pan-Methyl-Histone H3 (Lys9) Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, methylated-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 methylated at Lys9.* Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Pan-Methyl-Histone H3 (Lys9) Sandwich ELISA Kit #7864 detects endogenous levels of histone H3 when methylated at Lys9. As shown in Figure 1 using the Pan-Methyl-Histone H3 (Lys9) Sandwich ELISA Kit #7864, a high level of methylation at Lys9 is detected on Histone H3 in NIH/3T3 cells. These levels are unchanged in response to TSA-treatment. The level of total histone H3 (modified and unmodified) remains unchanged as shown by Western analysis (Figure 1). Similar results are obtained when COS and Jurkat cells are treated with TSA (data not shown). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Note: For this assay, it is recommended that lysates be thoroughly sonicated to ensure complete extraction of Histone H3 and an accurate absorbance reading.

ELISA - Western correlation

ELISA - Western correlation

Figure 1. Treatment of NIH/3T3 cells with trichostatin A (TSA) increases the acetylation of Histone H3 at Lys 9, detected by PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit #7121, and the di-methylation of Histone H3 at Lys4, detected byPathScan® Di-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit #7124. However, TSA treatment does not affect the level of general methylation at Lys9, detected by PathScan® Pan-Methyl-Histone H3 (Lys9) Sandwich ELISA Kit #7864, or the level of total Histone H3, detected by Pathscan® Total Histone H3 Sandwich ELISA Kit #7253. NIH/3T3 cells (70-80% confluent) were treated for 16-18 hours with 0.4 μM TSA at 37ºC. Absorbance readings at 450 nm are shown in the top figure while the corresponding Western blots using Histone H3 Antibody #9715 (panel A), Acetyl-Histone H3 (Lys9) Antibody #9671 (panel B), Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb #9725 (panel C) or Pan-Methyl-Histone H3 (Lys9) Antibody #4069 (panel D) are shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2. The relationship between the protein concentration of the lysate from untreated NIH/3T3 cells and the absorbance at 450 nm is shown.

Specificity

Specificity

Figure 3. Kit specificity as demonstrated by Western analysis of the ELISA microwell captured protein. Lysates were prepared from NIH/3T3 cells and incubated in microwells coated with the Pan-Methyl-Histone H3 (Lys9) capture antibody. Wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western analysis of NIH/3T3 cell starting lysate (lanes 1 & 2) and the captured protein (lane 3) was performed using Histone H3 Antibody #9715. The major band detected in the captured material corresponds to Histone H3 methylated at Lys9 (lane 3).


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.

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