Biology Reference
In-Depth Information
7.
To the second tube, add 0.75
m
l GTP for control, if needed.
8.
Wrap dye-GTP tube in aluminum foil to keep light out.
9.
Incubate both tubes at 37
C for 30 min.
10.
Add 0.5
m
l paclitaxel to each, mix well with pipette (40
m
M paclitaxel final).
11.
Incubate both tubes at 37
C for 20 min.
12.
During last incubation, add 100
m
lAB
sucrose to two 1.5-ml microcentrifuge
tubes, warm to room temperature, and add 1
m
l paclitaxel, mixing well.
13.
Very carefully layer MTs on top of sucrose cushion with pipette.
14.
Spin at room temperature at 14k RPM in microcentrifuge for 20 min.
15.
Carefully aspirate supernatant, changing tips often to reduce residual dye. Once
dye is gone, before cushion is gone, rinse cushion once with AB and then
aspirate cushion as well.
16.
Gently rinse pellet with 50
m
l room temperature AB
þ
þ
paclitaxel.
17.
Resuspend pellet in 50
m
l room temperature,
5 mg/ml final concentration.
18.
Store, light protected, at room temperature for up to 2 weeks.
If fluorophore density on the surface is too low (one or two fluorophores per micro-
tubule) or too high (individual fluorophores cannot be resolved), adjust fluorescent
GTP concentration in step 6.
2.2.2
Microtubule gliding assay
The microtubule gliding assay requires stock solutions prepared on the day of the
experiment, as described below. These stock solutions will generally keep on ice
for that day but should be discarded at the end of the day. Of particular concern
are solutions containing DTT and ATP. Finally, the solutions should all be warmed
to room temperature before using in the gliding assay since microtubules depolymer-
ize more rapidly at cold temperatures.
AB
þ
2 mM DTT:
1 ml AB, 10 ml DTT.
Use as AB
Biotin-LC-BSA:
70 ml, 1 mg/ml in AB (35 ml each of bio-BSA stock and AB)
BSA:
700 ml, 1 mg/ml in AB (694 mlABþ6.0 ml BSA stock)
SA:
60 ml, 0.5 mg/ml in AB (57 mlABþ3 ml SA stock)
CBAB:
375 ml, a-casein 1 mg/ml, BSA 1 mg/ml in AB (300 ml BSA and
75 ml a-casein stock)
Fluorescence anti-
bleach (FAB):
100 ml CBABþ1/3 ml oxygen scavenger, 1 ml b-ME, 1 ml
paclitaxel, 2.5 ml glucose (add last)
In preparing for the gliding assay experiment, we prepare slides with a sequential
coating from the glass slide up as follows. We begin with precleaned 24
60 mm
microscope glass slides and create flow-lanes on the slides using either vacuum
grease or double-sided tape. We cover these flow-lanes with 22
22 mmmicroscope
cover slips. The ends of the lanes are left open so that new solutions can be washed
