Microcon ultracentrifugal filter

K420 aliquot was thawed, exchanged into EM buffer through gel filtration with a Superose 6 10/300 GL column (GE Healthcare) and concentrated with Microcon ultracentrifugal filter (Merck Millipore, Billerica, Massachusetts). Concentrations of K420 and microtubules are determined by UV absorption. 37 μg of microtubules and 12.5 μg of K420 were mixed and diluted two-fold with water. The microtubule-K420 mix was then pelleted (13 K RPM, 24°C, 8 min) and resuspended in 5 μl two-fold diluted EM buffer. 0.7 μl of the resuspended mix was supplemented with 0.4 μl of 20 mM ATP, 0.4 μl of 20 mM AlCl3, 0.4 μl of 100 mM NaF and 2.1 μl of 4 mM MgCl2 to form a 4 μl-grid droplet and applied to Quantifoil holey carbon grids with 1.2 μm hole diameter and 1.3 μm spacing (Quantifoil Micro Tools GmbH, Großlöbichau, Germany) without glow discharge. The sample was allowed to incubate on the grid for 1 min and then most of the buffer was wicked away by touching the grid edgewise with a piece of filter paper. The grid was then mounted on a home-built plunge-freezing apparatus, blotted completely and plunged into liquid ethane.

The wild-type, monomeric human K349 construct was bacterially expressed and purified
as described (Kull et al., 1996 (link)), and 15%
(wt/vol) sucrose was added before snap freezing in liquid nitrogen and storing at
−80°C. A plasmid for the mutant N255K construct was generated from the
wild-type plasmid using the QuikChange site-directed mutagenesis kit (Agilent
Technologies; Santa Clara, CA). Thawed K349 (either wild-type or N255K) was exchanged
into EM buffer (25 mM PIPES, 25 mM KCl, 1 mM EGTA, 1 mM DTT) using three rounds of
dilution and concentration in a Microcon ultracentrifugal filter (EMD Millipore;
Billerica, MA). Microtubule batches were grown from 250 µg of lyophilized bovine
brain tubulin (Cytoskeleton; Denver, CO), resuspended in 25 µl EM buffer and
clarified (Beckman TLA 120.2, 100K RPM, 4°C) prior to incubation at 37°C.
Taxol (2 mM in DMSO) was added to equimolar levels with tubulin after 10 min of
incubation. Following ∼45 min of polymerization, microtubules were brought to
room temperature and a ∼twofold excess of K349 was added prior to pelleting
through a glycerol cushion (50 µl of EM buffer + 60% glycerol wt/vol +
200 µM taxol) in order to remove unbound motor and unpolymerized tubulin (20
min, Beckman TLA 120.2, 50K RPM, 24°C). The motor–microtubule complex was
resuspended in ∼10 µl of EM buffer plus 200 µM taxol.