Proteins to be used in methyltransferase assays were expressed in 293T cells by transient transfection and purified by above procedures. In all, 5 μl of input and purified protein was visualized by immunoblotting to ensure proper protein expression before use in methyltransferase assays. Prior to incubation with purified protein from human cells, the tRNA was first refolded by initial denaturation in 5 mM TRIS pH 7.5 and 0.16 mM EDTA and heated to 95 °C for two minutes before a two-minute incubation on ice. Refolding was conducted at 37 °C for 20 min in the presence of HEPES pH 7.5, MgCl2, and NaCl.
