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FIGURE 2:. Purification of S. frugiperda (SF+) tubulin. (A) Samples throughout the purification were analyzed by SDS–PAGE and stained with Coomassie blue. The lanes are as follows: crude extract (Crude), high-speed extract (Cleared), TOG column flow through (Flow), eluate, desalted eluate, concentrated eluate (conc.). (B) The crude extract, cleared extract, and TOG column flowthrough were transferred to nitrocellulose and probed with DM1 α-tubulin antibody. Tubulin in the extract was >95% depleted by a single pass over the TOG column. (C) Polymerization and sedimentation of SF+ tubulin. Tubulin was polymerized in the presence of GTP and glycerol and the monomer and polymer pools separated by sedimentation over glycerol cushions. Left lanes, total assembly reaction (Total), supernatant (Sup), and pellet. Right lanes, supernatant (Sup), pellet, and combined supernatant and pellet. (D) SF+ tubulin was polymerized in GTP and glycerol, stabilized with Taxol, deposited on formvar-coated grids, and negatively stained. Images are at 39,000× and 6600× magnification. Image published in: Widlund PO et al. (2012) © 2012 Widlund et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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