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 by
PathScan® Di-Methyl-Histone H3 (Lys4) Sandwich ELISA Kit #7124. However, TSA treatment does not affect the level of di-methylation at Lys9, detected by PathScan® Di-Methyl-Histone H3 (Lys9) Sandwich ELISA Kit #7862, 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 Di-Methyl-Histone H3 (Lys9) Antibody #9753 (panel D) are shown in the bottom figure.
Figure 2. The relationship between the protein concentration of the lysate from untreated NIH/3T3 cells and the absorbance at 450 nm is shown.
Figure 3. Kit specificity as demonstrated by Western blot analysis of the ELISA microwell captured protein. Lysates were prepared from NIH/3T3 cells and incubated in microwells coated with the Di-Methyl-Histone H3 (Lys9) capture antibody. Wells were washed, and the captured protein was solubilized in SDS gel loading buffer. Western blot 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 di-methylated at Lys9 (lane 3).
The PathScan® Di-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 di-methylated at Lys9. A Di-Methyl-Histone H3 (Lys9) Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, di-methyl-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 di-methylated at Lys9.
* Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Di-Methyl-Histone H3 (Lys9) Sandwich ELISA Kit #7862 detects endogenous levels of histone H3 when di-methylated at Lys9. As shown in Figure 1 using the Di-Methyl-Histone H3 (Lys9) Sandwich ELISA Kit #7862, a high level of di-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.
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).
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