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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 (20 mM phosphate buffer pH 7.4, 0.05% Tween-20, 20 µM S-adenosylmethionine, and 50-200 ng enzyme complex) add to the wells coated with the substrate. Incubate for 1 hr. Add antibody against methylated K27 residue of histone H3, incubate 1 hr. Then, add secondary HRP-labeled antibody and incubate 30 min. Finally, add HRP chemiluminescent substrates and read luminesence.
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, Human AEBP2 (NM_153207), a.a. 2-end, with N-terminal His-tag, MW= 53 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: 40 mM Tris-HCl, pH 8.0, 110 mM NaCl, 2.2 mM KCl, 200 mM imidazole, and 20% glycerol.
Genbank: NM_004456 (EZH2), NM_003797(EED), NM_015355 (SUZ12), NM_153207(AEBP2), NM_005610(RbAP48)
Host Cell Line: Sf9 cells
Storage Stability: At least 6 months at -80°C.
Tags: N-terminal FLAG-tag(EED), N-terminal His-tag (other components)
Uniprot: Q15910
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
References: 1. Morin, R., et al. (2010). Nature Genetics 42, 181 - 185.
2. 2. Varambally, S. et al. (2008). Science 322 (5908), 1695-1699.
3. Rakotobe, D. et al. (2008). Virol. J. 5, 32.
Application Reference(s): 1. Arcand, M., et al., ""Development of High-Throughput Assays to Study Methylases, Demethylases and Deacetylases."" Poster presented as part of the BioTechniques Fall 2011 Epigenetics Meeting.
2. Yap, D.B, et al., "Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethlyation." (2010). Blood. 117: 2451-2459.
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