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Microtubule polymerization assay

VK Viktoryia Kolas
JB Jose Sandino A. Bandonil
NW Niaz Wali
KH Kuo-Chiang Hsia
JS Jiun-Jie Shie
BC Bon-chu Chung
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For each steroid treatment, a reaction mixture was made from fluorescent porcine tubulin seed (4 μg/μL porcine tubulin, 833 μM GMPCPP, 333 ng/μL rhodamine tubulin), 30.4 ng FLAG-CLIP-170 and 70 nM steroid in BD buffer. Polymerization was induced at 37 °C for 10 min; premature polymerization was prevented through a short cold incubation (6 min, 4 °C). Subsequent treatment with 0.9% glutaraldehyde (3 min) was used to quench the reaction, and was in turn quenched with 100 μM Tris pH 7. The resulting reaction mixture was spun down on top of a 30% glycerol cushion (75,000 rpm, 17 min, 25 °C) to obtain polymerized tubulin from the pellet. The pellet was resuspended in BD buffer with taxol, and mounted for total internal reflection fluorescence (TIRF) microscopy with Revolution WD. Microtubules were visualized through a 561 nm filter, and imaged at 100X magnification. Microtubules were quatified using an Integrated Morphometry Analysis module in Metamorph.

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