EZH2 Chemiluminescent Assay Kit
The EZH2 Chemiluminescence Assay Kit is chemiluminescence-based assay kit designed to measure activity of the EZH2 (enhancer of zeste homolog 2) complex (EZH2/EED/SUZ12/RbAp48/AEBP) for screening and profiling purposes. The EZH2 Assay Kit comes in a convenient format, with a 96-well plate precoated with histone H3 peptide substrate, and enough antibody against methylated K27 residue of Histone H3, secondary HRP-labeled antibody, S-adenosylmethionine, methyltransferase assay buffer, and purified EZH2 complex for 100 enzyme reactions.
EZH2 Activity Assay Kit uses a highly specific antibody. In a first step, S-adenosylmethionine is incubated with methyltransferase and sample for one hour. This is followed by addition of the primary antibody. Finally, the plate is treated with an HRP-labeled secondary antibody and addition of the HRP substrate to produce chemiluminescence signal proportional to the EZH2 activity.
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- TBST buffer (1 x TBS, pH 8.0, containing 0.05% Tween20)
- Luminometer or microplate reader capable of reading chemiluminescence
- Rotating or rocker platform
| Catalog # | Component | Amount | Storage |
| 51004 | EZH2/EED/SUZ12/RbAp48/ AEBP2, FLAG-Tag, His-Tag* | 25 µg | -80°C |
| 52120 | 400 μM S-adenosylmethionine | 250 µl | -80°C |
| 52140F | Primary Antibody 6 | 12.5 µl | -80°C |
| 52131H | Secondary HRP-labeled Antibody 2 | 10 µl | -80°C |
| 52170 | 4x HMT Assay Buffer 2 | 3 ml | -20°C |
| 79556 | Blocking Buffer 1 | 50 ml | +4°C |
| HRP Chemiluminescent Substrate A | 6 ml | +4°C | |
| HRP Chemiluminescent Substrate B | 6 ml | +4°C | |
| 96-well plate precoated with histone substrate | 1 plate | +4°C |
*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.
EZH2 (enhancer of zeste homolog 2) is a histone-lysine N-methyltransferase enzyme, that acts by adding methyl groups to the lysine 27 (K27) of histone H3, making it a silent chromatin. It is a functional unit of the larger complex PRC2 (polycomb repressive complex 2), which also includes EED, Suz12, AEBP2 (adipocyte enhancer-binding protein) and RbAp48 (histone-binding protein RBBP4). PRC2 is crucial for epigenetic regulation and is involved in stem cell differentiation and embryonic development. Abnormalities in PRC2 result in cancer since this complex also promotes double strand DNA repair. EZH2 is seen as an attractive target in cancer therapy, as its levels are elevated in multiple cancer types (example, breast, renal cancer, melanoma, and lymphoma). The development of inhibitors for EZH2 and PRC2 is a promising area of research for the treatment of cancer.
Dillon S.C., et al. 2005. Genome Biology 6:227.
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