FIG. 7.

In vitro coprecipitation of microtubules and tubulin heterodimers with MP. Ni²⁺-NTA beads conjugated with MP (+ MP) or without MP (− MP) were incubated with in vitro-polymerized microtubules (polymer) or tubulin dimers (dimers). Pellet (P) and supernatant (S) fractions were separated by centrifugation and analyzed by SDS-PAGE and Western blotting with antibodies specific for MP and α-tubulin, respectively. (A) Immunodetection of MP in pellet but not in supernatant fractions, indicating that MP remained coupled to the Ni²⁺-NTA beads throughout the course of the binding reaction. (B) Immunodetection of α-tubulin in pellet and supernatant fractions. In pellet fractions containing MP, detectable amounts of both tubulin polymer and tubulin dimers were observed. In the absence of MP, no tubulin was detected in the pellets, indicating that tubulin cosedimentation with Ni²⁺-NTA beads occurred because of an interaction with MP.