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

Citation
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
Citations number
16
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Plant Sciences","Animal & Plant Sciences
Journal title
PLANT JOURNAL
ISSN journal
0960-7412 → ACNP
Volume
28
Issue
1
Year of publication
2001
Pages
117 - 122
Database
ISI
SICI code
0960-7412(200110)28:1<117:'CARMF>2.0.ZU;2-Z
Abstract
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.