'Caged cytoskeletons': a rapid method for the isolation of microtubule-associated proteins from synchronized plant suspension cells

McCutcheon, S Hemsley, RJ Jopson, MF Lloyd, CW

S. Mccutcheon et al., 'Caged cytoskeletons': a rapid method for the isolation of microtubule-associated proteins from synchronized plant suspension cells, PLANT J, 28(1), 2001, pp. 117-122

16

INGLESE

Article

Plant Sciences","Animal & Plant Sciences

PLANT JOURNAL

0960-7412 → ACNP

28

1

2001

117 - 122

ISI

0960-7412(200110)28:1<117:'CARMF>2.0.ZU;2-Z

In the cytoskeleton method for isolating microtubule-associated proteins MA P65, DcKRP120-1 and DcKRP120-2, carrot cells are first converted to protopl asts but this method cannot be used to isolate mitotic MAPS as mitotic sync hrony is eroded during lengthy cellulase treatment. Anti-microtubule cycle blocks would also be unsuitable. We report here a method for overcoming the se problems. Cellulase degradation of tobacco BY-2 cells for only several m inutes allows extraction of detergent-soluble proteins, leaving synchronize d 'caged cytoskeletons' for depolymerization and enabling affinity purifica tion of MAPs on neurotubules. This rapid and simple method should be of gen eral utility: it can be bulked up, avoids anti-microtubule blocks, and is a pplicable to other cell suspensions. The effectiveness of the caged cytoske leton 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.