'Caged cytoskeletons': a rapid method for the isolation of microtubule-associated proteins from synchronized plant suspension cells |
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Authors: | S. McCutcheon R. J. Hemsley M. F. Jopson C. W. Lloyd |
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Affiliation: | Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, UK. |
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Abstract: | 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. |
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Keywords: | tobacco BY-2 cytoskeleton microtubules microtubule-associated proteins cell synchrony. |
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