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 tri-methylation at Lys27, detected by PathScan® Tri-Methyl-Histone H3 (Lys27) Sandwich ELISA Kit #7866, 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 Tri-Methyl-Histone H3 (Lys27) Antibody #9756 (panel D) are shown in the bottom figure.Learn more about how we get our images
Figure 2. The relationship between the protein concentration of the lysate from HeLa cells and the absorbance at 450 nm is shown.Learn more about how we get our images
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 Tri-Methyl-Histone H3 (Lys27) 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 tri-methylated at Lys27 (lane 3).Learn more about how we get our images
|Product Includes||Volume||Solution Color|
|Histone H3 Rabbit mAb Coated Microwells||96 tests|
|Tri-Methyl-Histone H3 (Lys27) Rabbit Detection mAb (Biotinylated)||1 ea||Green (Lyophilized)|
|HRP-Linked Streptavidin (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml||Colorless|
|STOP Solution 7002||11 ml||Colorless|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml||Yellowish|
NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation.
Add 100 µl of STOP solution to each well. Shake gently for a few seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution.
posted June 2005
revised November 2013
The PathScan® Tri-Methyl-Histone H3 (Lys27) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of histone H3 when tri-methylated at Lys27. A Histone H3 Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, histone H3 is captured by the coated antibody. Following extensive washing, biotinylated Tri-Methyl Histone H3 (Lys27) Rabbit Antibody is added to detect the captured 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 tri-methylated at Lys27.
Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Tri-Methyl-Histone H3 (Lys27) Sandwich ELISA Kit #7866 detects endogenous levels of histone H3 when tri-methylated at Lys27. As shown in Figure 1 using the Tri-Methyl-Histone H3 (Lys27) Sandwich ELISA Kit #7866, a high level of tri-methylation at Lys27 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). 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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. PathScan is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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