Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Acetyl-Histone H2A Sandwich ELISA Kit #7233

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Important Ordering Details: Product is assembled upon order to ensure maximum activity. Domestic: Please allow up to two weeks for your order to be processed and shipped. International: Please allow up to three weeks, depending on the country, for your order to be processed and shipped.

Kit Includes Volume Solution Color
Histone H2A Ab CoatedMicrowells 96 tests
Acetylated-Lysine Detection Ab 11 ml Green
Anti-rabbit IgG, HRP- linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml 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, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M Mk

Reactivity Key:  H=Human  M=Mouse  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

CST's PathScan® Acetyl-Histone H2A Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of acetylated lysines on Histone H2A. A Histone H2A Antibody has been coated onto the microwells. After incubation with cell lysates, Histone H2A is captured by the coated antibody. Following extensive washing, Acetylated-Lysine Rabbit mAb (Ac-K2-100) is added to detect the acetylated lysines on the Histone H2A protein. Anti-rabbit IgG, HRP linked Antibody 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 H2A.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Acetyl-Histone H2A Sandwich ELISA Kit detects endogenous levels of Acetylated Histone H2A. Using this Sandwich ELISA Kit #7233, acetylated lysines on Histone H2A are detected when treated with TSA in Jurkat cells. However, the levels of Histone H2A remain unchanged, as shown by Western analysis using the Histone H2A Antibody #2578 (figure 1). COS and NIH 3T3 cells treated with TSA show similar results (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.

Sandwich ELISA

Sandwich ELISA

Figure 1: Treatment of Jurkat cells with TSA causes accumulation of acetylation on Histone H2A, detected by Sandwich ELISA Kit, #7233, but does not affect the level of total Histone H2A protein, detected by Western analysis. OD 450nm readings are shown in the top figure, while the corresponding Western blot using the Acetylated Lysine mouse mAb (Ac-K-103) #9681 (left panel) or Histone H2A Antibody #2578 (right panel), is shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of lysates from untreated and TSA treated COS cells and kit assay optical density readings. COS cells were treated with TSA (4 µM overnight). An acid extraction was performed for cell lysis in the presence of 5 mM sodium butyrate.

Background

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).

  1. Workman, J.L. and Kingston, R.E. (1998) Annu Rev Biochem 67, 545-79.
  2. Hansen, J.C. et al. (1998) Biochemistry 37, 17637-41.
  3. Strahl, B.D. and Allis, C.D. (2000) Nature 403, 41-5.
  4. Cheung, P. et al. (2000) Cell 103, 263-71.
  5. Bernstein, B.E. and Schreiber, S.L. (2002) Chem Biol 9, 1167-73.
  6. Jaskelioff, M. and Peterson, C.L. (2003) Nat Cell Biol 5, 395-9.
  7. Thorne, A.W. et al. (1990) Eur J Biochem 193, 701-13.
  8. Hendzel, M.J. et al. (1997) Chromosoma 106, 348-60.
  9. Goto, H. et al. (1999) J Biol Chem 274, 25543-9.
  10. Preuss, U. et al. (2003) Nucleic Acids Res 31, 878-85.
  11. Dai, J. et al. (2005) Genes Dev 19, 472-88.

Application References

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