Biology Reference
In-Depth Information
FIGURE 14.3
Polymerization timecourses monitored by the change DAPI fluorescence, performed as
described in the text. Solutions of bovine brain tubulin (8
M), and GTP
(1 mM) in PME buffer were incubated with varying concentrations of podophyllotoxin (1
m
M), DAPI (10
m
m
M
(
-)) at room temperature for 35 min. The
control (—) had no podophyllotoxin. The multiwell plate was incubated at 37
C in the
SynergyMx microplate reader for 5 min, after which the fluorescence at 450 nm (360 nm
excitation) was recorded to establish a baseline. Microtubule assembly was then induced by
paclitaxel in DMSO to a final concentration of 8
); 2
m
M (----); 4
m
M(-
-
-); 6
m
M (---); 8
m
M(-
-
M. No data were collected in the interval
between 5 and 10 min because the assay plate was removed for addition of paclitaxel during
that period. The final concentration of DMSO in each sample was 8% (v/v).
m
14.1.5
Measurement of drug effects—Polymerization inhibitors
The effect of MT-destabilizing, polymerization-inhibiting drugs can be measured by
following the inhibition of polymerization as a function of drug concentration. This
is illustrated in
Fig. 14.3
using DAPI fluorescence to monitor MT polymerization.
14.2
MATERIALS AND EQUIPMENT
There are three components of the experiment that require decisions: (1) the buffer,
(2) the protein, and (3) the instrument. We will discuss these in sequence.
-
The buffer used here is based on Na Pipes, but Na glutamate is a very attractive
alternative for turbidity assay since it allows the reaction to be “tuned” simply by
changing the concentration of glutamate (
Hamel, 2003
). In addition, glutamate
promotes production of sheet polymers as well as MT, and these produce higher
OD per milligram polymer than do MT, increasing the signal of polymerization.
Often polymerization-promoting compounds are included in the buffer, as
discussed in
Section 1.3
. Here, we suggest Pipes/Mg/TMAO because it produces a
