Product Pathways - PathScan ELISA
PathScan® Acetyl-Histone H2B (Lys20) Sandwich ELISA Kit #7222
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Acetyl-Histone H2B (Lys20) Detection Ab | 11 milliliters | Green |
| Histone H2B Antibody Coated Microwells | 96 tests | |
| HRP-Linked Streptavidin | 11 milliliters | Red |
| TMB Substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| Sealing Tape | 2 sheets | |
| 20X Wash Buffer | 25 milliliters | Colorless |
| Sample Diluent | 25 milliliters | Blue |
| Cell Lysis Buffer (10X) # 9803 | 15 milliliters | 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 (10X), which is stored at –20°C (packaged separately).
Species Cross-Reactivity
H M Mk
Reactivity Key: H=Human M=Mouse Mk=Monkey
Description
The PathScan® Acetyl-Histone H2B (Lys20) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Histone H2B when acetylated at Lys20. A Histone H2B Antibody* has been coated onto the microwells. After incubation with cell lysates, histone H2B protein (acetylated and non-acetylated) is captured by the coated antibody. Following extensive washing, biotinylated Acetyl-Histone H2B (Lys20) Antibody* is added to detect acetylated Lys20 on the histone H2B 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 H2B acetylated at Lys20.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Acetyl-Histone H2B (Lys20) Sandwich ELISA Kit detects endogenous levels of histone H2B when acetylated at Lys20. As shown in Figure 1 using the Acetyl-Histone H2B (Lys20) Sandwich ELISA Kit #7222, a high level of acetylation at Lys20 on histone H2B is detected in NIH/3T3 cells when treated with trichostatin A (TSA). The level of total histone H2B (acetylated and non-acetylated) 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).
ELISA
Figure 2. The relationship between the protein concentration of lysates from untreated and TSA-treated NIH/3T3 cells (see the legend of Figure 1) and the absorbance at 450 nm.
Sandwich ELISA
Figure 1. Treatment of NIH/3T3 cells with trichostatin A (TSA) increases the acetylation of histone H2B at Lys20 detected by PathScan® Acetyl-Histone H2B (Lys20) Sandwich ELISA Kit #7222. TSA treatment does not affect the level of histone H2B that is detected by Western analysis. 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 H2B Antibody #2722 (left panel) or Acetyl-Histone H2B (Lys20) Antibody #2571 (right panel) are shown in the bottom figure.
Background
Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of four core histone proteins (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 and 23 (2,3). 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).
- Workman, J.L. and Kingston, R.E. (1998) Annu. Rev. Biochem. 67, 545-579.
- Hansen, J.C. et al. (1998) Biochemistry 37, 17637-17641.
- Strahl, B.D. and Allis, C.D. (2000) Nature 403, 41-45.
- Cheung, P. et al. (2000) Cell 103, 263-271.
- Bernstein, B.E. and Schreiber, S.L. (2002) Chem. Biol. 9, 1167-1173.
- Jaskelioff, M. and Peterson, C.L. (2003) Nat. Cell Biol. 5, 395-399.
- Thorne, A.W. et al. (1990) Eur. J. Biochem. 193, 701-713.
- Hendzel, M.J. et al. (1997) Chromosoma 106, 348-360.
- Goto, H. et al. (1999) J. Biol. Chem. 274, 25543-25549.
- Preuss, U. et al. (2003) Nucleic Acids Res. 31, 878-885.
- Dai, J. et al. (2005) Genes Dev. 19, 472-488.
Application References
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