PathScan Sandwich ELISA Kits

PathScan® Di-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit #7124

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Applications Key:
Reactivity Key: H = Human  M = Mouse  Mk = Monkey  
Species cross-reactivity is determined by Western blot.

Protocols

7124

Sandwich ELISA,

Specificity / Sensitivity

CST's PathScan® Di-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit #7124 detects endogenous levels of histone H3 when di-methylated at Lys4. As shown in Figure 1 using the Di-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit #7124, a high level of di-methylation at Lys4 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).

Description

The PathScan® Di-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of histone H3 when di-methylated at Lys4. A Di-Methyl-Histone H3 (Lys4) Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, di-methyl-histone H3 (Lys4) 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 di-methylated at Lys4.
* Antibodies in kit are custom formulations specific to kit.

ELISA - Western correlation

Figure 1. Treatment of COS cells with trichostatin A (TSA) increases the di-methylation of Histone H3 at Lys4 detected by PathScan® Di-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit #7124. 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 37ºC. Absorbance readings at 450 nm are shown in the top figure while the corresponding Western blots using Histone H3 Antibody #9715 (left panel) or Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb #9725 (right panel) are shown in the bottom figure.

Sensitivity

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 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, 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, 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 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. et al. (1998) Annu Rev Biochem67, 545 - 79.
2. Hansen, J.C. et al. (1998) Biochemistry37, 17637 - 41.
3. Strahl, B.D. and Allis, C.D. et al. (2000) Nature403, 41 - 5.
4. Cheung, P. et al. (2000) Cell103, 263 - 71.
5. Bernstein, B.E. and Schreiber, S.L. et al. (2002) Chem Biol9, 1167 - 73.
6. Jaskelioff, M. and Peterson, C.L. et al. (2003) Nat Cell Biol5, 395 - 9.
7. Thorne, A.W. et al. (1990) Eur J Biochem193, 701 - 13.
8. Hendzel, M.J. et al. (1997) Chromosoma106, 348 - 60.
9. Goto, H. et al. (1999) J Biol Chem274, 25543 - 9.
10. Preuss, U. et al. (2003) Nucleic Acids Res31, 878 - 85.
11. Dai, J. et al. (2005) Genes Dev19, 472 - 88.

Application References

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Companion Products

• 9717 Histone H3 (3H1) Rabbit mAb
• 9725 Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb
• 7121 PathScan® Acetyl-Histone H3 (Lys9) Sandwich ELISA Kit
• 9726 Di-Methyl-Histone H3 (Lys4) Antibody
• 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
• 7232 PathScan® Acetylated Histone H3 Sandwich ELISA Kit
• 7123 PathScan® Mono-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit
• 7125 PathScan® Tri-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit
• 9808 Phosphate Buffered Saline (PBS-20X)
• 7122 PathScan® Acetyl-Histone H3 (Lys18) Sandwich ELISA Kit
• 7004 TMB Substrate
• 7002 STOP Solution
• 9715 Histone H3 Antibody

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.