MLL3/WARD complex of human MLL3 (GenBank Accession No. NM_170606) a.a. 4689-4911(end) with N-terminal His-tag and MW= 27 kDa, human WDR5 (GenBank Accession No. NM_017588), a.a. 2-334(end ) with N-terminal His-tag and MW= 37 kDa, human Ash2L (GenBank Accession No. NM_001105214), a.a. 2-534(end) with N-terminal His-tag and MW= 61 kDa, human RbBP5 (GenBank Accession No. NM_005057), a.a. 2-538(end) with N-terminal His-tag and MW= 60 kDa, and human DPY30 (GenBank Accession No. NM_032574) a.a. 2-99(end) with N-terminal His-tag and MW=12 kDa individually expressed in an E. coli expression system.
1. Patel, A., et al., J. Biol. Chem. 2009; 284(36): 24242-24256. 2. Mohan, M., et al., Nat. Rev. Cancer 2010; 10(10): 721-728.
Storage/Stability
At least 6 months at -80°C.
Assay Conditions
H3, 1-21-Biotin was coated onto a neutravadin plate at 0.4 μg/ml. Reaction mixture contained 1X HMT buffer 1 (50 mM Tris buffer pH 8.8, 5 mM MgCl2, 4 mM DTT, 0.05% Tween-20), various amounts of S-adenosylmethionine (40 μM for SA determination, 20 μM for inhibition), and/or various amounts of MLL3 Complex—incubated for 1-hour at room temperature. Followed by 1-hour incubation with primary antibody against 3MeK4 Histone 3 (cell signaling #9751) and 30-minute incubation with α-rabbit secondary antibody (abcam). Luminescence was developed with ELISA ECL Substrates A and B (BPS #79670).
Instructions for Use
Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage. Avoid multiple freeze/thaw cycles.
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