RESUMEN
In the cytoskeleton method for isolating microtubule-associated proteins MAP65, DcKRP120-1 and DcKRP120-2, carrot cells are first converted to protoplasts but this method cannot be used to isolate mitotic MAPs as mitotic synchrony is eroded during lengthy cellulase treatment. Anti-microtubule cycle blocks would also be unsuitable. We report here a method for overcoming these problems. Cellulase degradation of tobacco BY-2 cells for only several minutes allows extraction of detergent-soluble proteins, leaving synchronized "caged cytoskeletons" for depolymerization and enabling affinity purification of MAPs on neurotubules. This rapid and simple method should be of general utility: it can be bulked up, avoids anti-microtubule blocks, and is applicable to other cell suspensions. The effectiveness of the caged cytoskeleton method is demonstrated by comparing known MAPs (the 65 kDa structural MAPs and the kinesin-related protein, TKRP125) in synchronized cells taken at the mitotic peak with those in unsynchronized cells.
