Analysis of the BUNV N/RNA complex using gel filtration and analytical ultracentrifugation. (A) Bacterially expressed His-tagged BUNV N protein was purified by nickel affinity chromatography, after which the His-tag was removed by thrombin cleavage and applied to a Superdex S300 size exclusion column at a flow rate of 1 mL/min. The elution profile of the N protein is shown in black, and the profile of known molecular weight standards applied to the same column is shown in gray. (B) Three different concentrations of the BUNV N protein were analyzed using analytical sedimentation centrifugation at 40,000 rpm for 14 h. Sedimentation was measured using absorbance optics at 260 nm, and the resulting data analyzed using SEDFIT as described in Materials and Methods. (C) N/RNA complexes at a concentration of 0.25 mg/mL were subjected to complete RNase A digestion, and analyzed by analytical sedimentation centrifugation alongside undigested samples. Sedimentation was measured and analyzed as described above.
