H tirf module

Paclitaxel-stabilized MTs were prepared containing 10% rhodamine tubulin, 10% biotinylated tubulin, and 80% unlabelled tubulin (Cytoskeleton, Inc.), then treated ± 0.1 mg/ml subtilisin for 30 min to remove predominantly β-tubulin C-terminal tails (CTTs), checked by western blot. The reaction was stopped by the addition of 10 mM pefabloc, and MTs were isolated by centrifugation. GFP-CKK domain binding was analyzed by TIRFM, using flow chambers assembled from plasma-cleaned glass coverslips and microscope slides. Chambers were incubated sequentially with 1 mg/ml PLL-PEG biotin (Susos AG), block solution (1% plurionic F-127, 4 mg/ml casein), 0.5 mg/ml neutravidin, and MTs ± CTTs (as indicated). Each incubation was followed by two washes with MRB80 buffer (80 mM PIPES, 4 mM MgCl2, and 1 mM EGTA [pH 6.8]) supplemented with 80 mM KCl, 20 μM paclitaxel, 4 mM DTT and 2 mg/ml casein. The final binding reaction contained 200 nM CKK-GFP in MRB80 with 80 mM KCl, 20 μM taxol, 4 mM DTT and 2 mg/ml casein and an oxygen scavenger mix (400 μg/ml glucose oxidase, 200 μg/ml catalase). TIRFM was performed on an Eclipse Ti-E inverted microscope with a Perfect Focus System, CFI Apo TIRF 1.49 N.A. oil objective, H-TIRF module and LU-N4 laser unit (Nikon). Images were recorded with a 100 ms exposure on an iXon DU888 Ultra EMCCD camera (Andor) controlled with NIS-Elements AR Software (Nikon).