Biology Reference
In-Depth Information
14.3.5.2
Material
1.
PME buffer (0.1 M Pipes, 1 mM MgSO
4
, 2 mM EGTA, pH 6.90).
2.
GTP stock solution (
65 mM in double-distilled water).
3.
DMSO.
4.
DAPI stock solution (
1.5 mM in double-distilled water).
5.
Paclitaxel stock solution (2 mM in DMSO); paclitaxel working solution (200
m
M
in DMSO).
6.
Ligand to be tested in DMSO.
7.
FLUOTRAC 200, 96-well microplate, black/clear bottom (Grenier Bio-One,
Germany).
14.3.5.3
Method
1.
Set the reading chamber of the plate reader to 37
C.
2.
Set the excitation at 360 nm and the emission at 450 nm.
3.
Prepare ligand solutions in DMSO. We prepare individual samples that are
25
, the desired final concentration. In this way, the same volume of ligand
solution is added to each well.
4.
Prepare tubulin stock solution that contains GTP and DAPI in PME buffer to the
following final concentrations: 8.7
m
m
M tubulin, 1.09 mM GTP, and 10.9
M
DAPI.
5.
Place 184
m
l tubulin stock solution in each well.
l of the ligand sample in DMSO to the well.
7.
Mix by stirring the solution with a P20 pipet tip while pipetting up and down.
Incubate the plate at room temperature to allow the ligand to bind to the protein.
We usually perform the incubation for about 30-40 min.
8.
Place the plate in the reading chamber and incubate for 5 min.
9.
Record the fluorescence at 450 nm to obtain a baseline.
10.
Remove the plate from the chamber.
11.
Add 8
6.
Add 8
m
l of the paclitaxel working solution in DMSO (to a final paclitaxel
concentration of 8
m
M) and mix carefully using the pipet. Try to avoid bubble
formation at this point.
12.
Return the plate to the plate reader.
13.
Monitor the fluorescenceat 450 nmover timeuntil a satisfactoryplateau is reached.
m
References
