McCutcheon S, Hemsley R J, Jopson M F, Lloyd C W
Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, UK.
Plant J. 2001 Oct;28(1):117-22. doi: 10.1046/j.1365-313x.2001.01134.x.
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.
在用于分离微管相关蛋白MAP65、DcKRP120 - 1和DcKRP120 - 2的细胞骨架方法中，胡萝卜细胞首先要转化为原生质体，但这种方法不能用于分离有丝分裂微管相关蛋白，因为在长时间的纤维素酶处理过程中有丝分裂同步性会受到破坏。抗微管周期阻断法也不合适。我们在此报告一种克服这些问题的方法。对烟草BY - 2细胞仅进行几分钟的纤维素酶降解，就能提取去污剂可溶的蛋白，留下同步化的“笼状细胞骨架”用于解聚，并能在神经微管上对微管相关蛋白进行亲和纯化。这种快速且简单的方法应具有广泛的实用性：它可以扩大规模，避免抗微管阻断，并且适用于其他细胞悬浮液。通过比较处于有丝分裂高峰期的同步化细胞与非同步化细胞中已知的微管相关蛋白（65 kDa结构微管相关蛋白和驱动蛋白相关蛋白TKRP125），证明了笼状细胞骨架方法的有效性。
