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Encompassing Amino Acids: 2-end (all components)
Applications: Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
Assay Conditions: 50 µl reaction mix (50 mM Tris pH 8.8, 5 mM MgCl2, 4 mM DTT, 20 µM S-adenosylmethionine, and 50-200 ng enzyme complex) add to the wells coated with the substrate. Incubate for 1 hour. Add antibody against methylated K27 residue of histone H3, incubate 1 hour. Then incubate 1 hour with the HRP-labeled secondary antibody, followed by addition of the HRP substrate to produce chemiluminescence.
Background: EZH2 is the catalytic subunit of the PRC2/EED-EZH2 complex, which methylates Lys9 (H3K9me) and Lys27 (H3K27me) of histone H3, leading to transcriptional repression of the affected target gene. Able to mono-, di- and trimethylate Lys27 of histone H3 to form H3K27me1, H3K27me2 and H3K27me3, respectively. Compared to EZH2-containing complexes, it is more abundant in embryonic stem cells and plays a major role in forming H3K27me3, which is required for embryonic stem cell identity and proper differentiation. The PRC2/EED-EZH2 complex may also serve as a recruiting platform for DNA methyltransferases, thereby linking two epigenetic repression systems. Genes repressed by the PRC2/EED-EZH2 complex include HOXC8, HOXA9, MYT1, CDKN2A and retinoic acid target genes. EZH2 can also methylate non-histone proteins such as the transcription factor GATA4.
Description: Complex of human EZH2 (GenBank Accession No. NM_004456), a.a. 2-end with N-terminal His-tag, MW= 86 kDa, human EED (NM_003797), a.a. 2-end with N-terminal FLAG-tag, MW= 51 kDa, human SUZ12 (NM_015355), a.a. 2-end with N-terminal His-tag, MW = 87 kDa, and human RbAp48 (NM_005610), a.a. 2-end with N-terminal His-tag, MW = 48 kDa, co-expressed in a baculovirus expression system.
Format: Aqueous buffer solution
Formulation: 25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.05% Tween-20, and 10% glycerol
Genbank: NM_004456 (EZH2), NM_003797(EED), NM_015355 (SUZ12), NM_005610(RbAP48)
Host Cell Line: Sf9 cells
Storage Stability: At least 6 months at -80°C.
Tags: N-terminal His-tag (SUZ12, EZH2, RbAP48), N-terminal FLAG-tag (EED)
Uniprot: Q15910
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
References: 1. Varambally, S. et al. Science 322 (5908), 1695-1699 (2008).
2. Rakotobe, D. et al., Virol. J. 5, 32 (2008).