Product Pathways - PathScan ELISA
PathScan® Acetyl-Histone H2B Sandwich ELISA Kit #7178
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Histone H2B Antibody Coated Microwells | 96 tests | |
| Acetylated-Lysine Detection Ab | 11 milliliters | Green |
| Anti-Mouse IgG HRP-Linked Ab | 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 Mk
Reactivity Key: H=Human Mk=Monkey
Description
The PathScan® Acetyl-Histone H2B Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of acetylated lysines on Histone H2B. The Histone H2B Antibody #2722* has been coated onto the microwells. After incubation with cell lysates, Histone H2B is captured by the coated antibody. Following extensive washing, Acetylated-Lysine Mouse mAb (Ac-K-103) #9681* is added to detect the acetylated lysines on the Histone H2B protein. Anti-mouse IgG, HRP linked Antibody #7076 is then used to recognize the bound detection antibody. HRP substrate, TMB is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of acetylated Histone H2B.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Acetyl-Histone H2B Sandwich ELISA Kit detects endogenous levels of acetylated Histone H2B. Using this Sandwich ELISA Kit #7178, acetylated lysines on Histone H2B are detected when treated with TSA in COS cells. However, the levels of total Histone H2B protein remain unchanged, as shown by Western blot analysis, using the Histone H2B Antibody #2722 (figure 1). Jurkat cells treated with TSA show similar results (data not shown).
Western Blotting
Figure 1: Treatment of COS cells with TSA causes accumulation of acetylation on Histone H2B, detected by Sandwich ELISA Kit #7178, but does not affect the level of total histone H2B protein, detected by Western analysis. OD 450 readings are shown in the top figure, while the corresponding Western blots using the Acetylated Lysine Mouse mAb (Ac-K-103) #9681 (left panel) or Histone H2B Antibody #2722 (right panel), are shown in the bottom figure.
ELISA
Figure 2: The relationship between protein concentration of lysates from untreated and TSA-treated Cos cells and kit assay optical density readings. COS cells (80% confluence) were treated with TSA (0.4 uM overnight) and then lysed.
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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