Fig. 5. The C-terminal fragment of XMAP215 has the highest affinity for pure tubulin microtubules. (A) GFP fusion proteins of FrN, FrM and FrC were incubated with pure tubulin microtubules and sedimented without fixation. CY5-labeled microtubule seeds are shown in a blue pseudocolor. Note that all three of the fragments studied are associated with microtubules whilst GFP alone does not bind to microtubules. (B) Microtubule spin-down assay. c-myc-tagged FrN, FrM and FrC were incubated with 1 µM pure tubulin microtubules. After sedimentation, pellets and supernatants were analyzed by SDS–electrophoresis followed by Coomassie Blue staining. BSA was present in reactions to prevent precipitation and/or absorption of XMAP215 fragments on the walls of test tubes. Note that the FrN band (62 kDa) is just under the BSA band. (C) Immunoblotting results of the same experiment shown in (B).