Biology Reference
In-Depth Information
Lyophilized KHC is resuspended at 5
m
g/
m
L in kinesin reconstitution buffer com-
posed of 100 mM PIPES pH 7.0, 200 mM KCl, 2 mM MgCl
2
, 1 mM DTT, 20
m
M
ATP. 0.5
m
L Aliquots are frozen in liquid nitrogen and stored at
80
C.
For use, one aliquot of kinesin is diluted at 0.5 mg/mL in PEM buffer containing
0.2 mg/mL casein (PEM-C) and 20
m
M ATP. Then kinesin is diluted at 10
m
g/mL in
PEM-C containing 100
m
M ATP.
Tips
Casein is used to avoid nonspecific interactions between microtubule seeds
or free tubulin with glass coverslips. It is poorly soluble at neutral pH, but
it can be rapidly dissolved after effective alkalinization with 10N KOH. After
complete solubilization, pH is set back to 6.9 using HCl.
10.3.2.4
Taxol-stabilized microtubule seeds polymerization
To prepare stabilized microtubule seeds as templates to nucleate microtubule
growth, we use an alternative method to GMPCPP-stabilized microtubule seeds.
Seeds are stabilized using Taxol for easiness and swiftness.
A 4 g/L mix of Cy5-labeled tubulin and nonlabeled tubulin (with a 1:10 ratio of
labeled to unlabeled tubulin) is incubated for 15 min at 37
C with 1 mM GTP and
10% DMSO. These conditions allow the spontaneous polymerization of microtubule
seeds. After incubation, microtubule seeds are diluted to 1:100 in PEM containing
10
m
M Taxol. These seeds can be stored for 2 days at room temperature.
10.3.2.5
Preparation of the incubation chambers
Sample chambers are prepared using standard glass microscopy coverslips. Cover-
slips are washed consecutively in 10N HCl, Nanopure water, and 90% ethanol and
air-dried. A 2- to 3-mm-width channel is delimited by two strips of adhesive tape on a
25-mm-diameter glass coverslip. A 12-mm-diameter coverslip is clamped on the
first one with two additional strips of tape. The solutions are perfused in the incuba-
tion chamber using a pipette and a filter paper to aspirate the solutions at the opposite
end of the channel. The incubation chamber is then placed in an Attofluor chamber
for observation under the microscope.
10.3.2.6
Binding of microtubule seeds and microtubule elongation
10.3.2.6.1
Nonspecific kinesin immobilization
The 10
m
g/mL kinesin solution is incubated for 5 min at room temperature in the
chamber to allow the adsorption of motor protein on glass. The chamber is then
washed three times with PEM containing 0.5 mg/mL casein, and this solution is in-
cubated for 5 min at room temperature to block the nonspecific adsorption of micro-
tubule seeds and of free tubulin added in the experiments. It also prevents kinesin-1
from denaturation (
Ozeki et al., 2009
).
