Figure 6.
Tubulin binding of Op18F and truncated derivatives. (A) Binding of tubulin (20 μM, 15 min at 37°C) to M2-coupled beads alone (lane a) or M2-coupled beads coated with Op18F (lanes b and c). Lane c shows that tubulin binding to Op18F-coated beads is blocked by addition of excess soluble Op18 (75 μM). Bead-bound material was separated by a rapid one-step procedure involving centrifugation through a sucrose gradient. Sedimented material was analyzed by SDS-PAGE followed by detection of proteins by Coomassie Blue staining. The position of of the heavy (HC) and light (LC) chains derived from the M2 antibody, as well as tubulin, Op18F, and the competitor Op18, are indicated. (B) Tubulin (20 μM) was added to Op18F coupled to M2 beads, and bead-bound tubulin was quantitated after incubation at 37°C for the indicated times. The molar ratio of tubulin associated with Op18 was determined as described in MATERIALS AND METHODS, and the contribution of nonspecific binding (∼8%) was subtracted from the presented data. The data points show the mean of duplicate samples, and the error bars reflect the range of duplicate samples. For most data points the error bars are smaller than the symbols. Data are representative of three independent experiments performed in duplicate.