Figure 1. Treatment of NIH/3T3 cells with trichostatin A (TSA) increases the acetylation of histone H3 at Lys9, 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 total histone H3 that is 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 (left panel), Acetyl-Histone H3 (Lys9) Antibody #9671 (middle panel) or Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAb #9725 (right panel) are shown in the bottom figure.
Figure 2. The relationship between the protein concentration of the lysate from untreated and TSA-treated NIH/3T3 cells and the absorbance at 450 nm is shown.
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 Histone H3 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 (lanes 3 & 4) was performed using Histone H3 Antibody #9715. The major band detected in the captured material corresponds to histone H3 (lanes 3 & 4).
|REACTIVITY||H M Mk|
|Product Includes||Volume||Solution Color|
|Histone H3 Rabbit mAb Coated Microwells||96 tests|
|Histone H3 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Total Histone H3 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of histone H3. A Histone H3 Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, histone H3 (modified and unmodified) is captured by the coated antibody. Following extensive washing, Histone H3 mouse mAb is added to detect the captured histone H3 protein. Anti-Mouse IgG, HRP-linked Antibody
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 total histone H3.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Total Histone H3 Sandwich ELISA Kit #7253 detects endogenous levels of histone H3. As shown in Figure 1, using the Total Histone H3 Sandwich ELISA Kit #7253, a high level of histone H3 is detected in NIH/3T3 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 COS and Jurkat cells are treated with TSA (data not shown). Note: For this assay, it is recommended that lysates be thoroughly sonicated to ensure complete extraction of histone H3 and an accurate absorbance reading. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Human, Mouse, Monkey
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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