Zonal velocity sedimentation. An aliquot (50 μg) of the tag-free wild-type MimiTgs protein was mixed with catalase (50 μg), BSA (50 μg), and cytochrome c (50 μg). The mixture was applied to a 5 mL 15%–30% glycerol gradient containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM DTT, 1 mM EDTA, 0.05% Triton X-100. The gradient was centrifuged at 50,000 rpm for 17 h at 4°C. Fractions (∼0.18 mL) were collected from the bottom of the tube. (A) Aliquots (20 μL) of the even-numbered gradient fractions were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown, with the bottom (heaviest) gradient fraction at left and the top (lightest) fraction at right. The identities of the polypeptides are indicated. (B) Aliquots (1 μL) of the even-numbered fractions were assayed for cap guanine-N2 methyltransferase activity. The extents of methyl transfer from 50 μM [3H-CH3]AdoMet to 1 mM m7GpppA are plotted.
