Figure 3.

Velocity sedimentation analysis of purified His-GlpF. OG-solubilized, affinity-purified His-GlpF was sedimented at 140,000 × g, for 18 hr at 20°C on a continuous gradient containing 5–20% sucrose. The gradient contained 20 mM Tris⋅HCl (pH 8.0), 5 mM EDTA, and 3% OG. (A) The sedimentation coefficient of GlpF was determined by comparison with multiple water-soluble standards. (B) Mobility of purified GlpF was determined in gradients also containing no additional salt, 300 mM NaCl, or 300 mM MgCl₂, or in 0.1% SDS. Twenty fractions were collected; top of gradient is on the left. Mobility of His-GlpF was determined by immunoblotting. (C) Fractions 6 and 11 from a gradient containing OG and 300 mM NaCl were analyzed by immunoblotting with anti-His or anti-HA monoclonal antibodies before (a) and after (b) a second affinity purification using Ni-NTA-agarose magnetic beads.