Biology Reference
In-Depth Information
9.
Quickly wipe the bottom of the plate, dry with a paper towel, and then again
with a Kimwipe under the wells to be read.
10.
Put the plate into the instrument and record OD (350 nm) at 20 s intervals for a
total of 20 min.
11.
(Optional). At the end of the recording, 50
l samples are removed from each
well and centrifuged as in the cuvette assay, and supernatant removed for
protein assay.
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14.3.4
Analysis of turbidity
The most important point to remember in interpreting turbidity is that turbidity does
not only arise from MTs, and that changes in turbidity yield may originate in changes
in polymer
type
as well as in polymer
amount
. For this reason, it can be very valuable to
measure the protein concentration on the supernatant of the samples taken and centri-
fuged at the end of the OD readings, as discussed in
Section 1.2
. If the OD at the end of
the readings differs between two samples, but the supernatant protein concentration is
the same, it may be concluded that the polymers in the two samples are not the same,
and further study, for example, electron microscopy, might be considered. Similarly,
differences in lag time or maximum slope may be clues to suggest further experiments.
Beyond this point, there are three parts of the timecourse that are useful in the
interpretation of turbidity assays of polymerization: the lag time, the rate of increase,
and the steady-state level of OD. As solution conditions more strongly favor poly-
merization (higher tubulin concentration, increased temperatures (up to 37
C), ad-
dition of polymerization promotors) the lag phase shortens, the rate of increase
steepens, and the final steady-state OD value increases. Detailed discussions of these
points may be found in
Gaskin et al. (1974), Correia and Williams (1983), Hall and
Minton (2005)
, and
Gaskin (2011)
.
14.3.5
Fluorescence assay in multiwell plates
14.3.5.1
Introduction
This protocol uses a final volume of 200
l in each well. Although it consumes more
tubulin than the previously described assay, it is easier for less-experienced experimen-
talist (such as an undergraduate student) to perform. Paclitaxel-induced assembly is
used because the critical concentration of tubulin in the presence of paclitaxel is very
low; therefore, almost all of the tubulin in each sample will assemble into MTs.
Nevertheless, this particular procedure uses about twice as much tubulin as the turbid-
ity assays described. The total volume in each well can be decreased by reducing
each solution volume proportionally. Note that, while this protocol uses a temperature
of 37
C, the polymer yield in the control would not be much different at lower tem-
perature (e.g., 30
C) as discussed in
Section 1.3
and shown in
Table 14.1
.
The protocol described below is to assay for inhibitors of MT assembly. All
buffer solutions contain DMSO at a final concentration of 4%. If ligands to be tested
are potential inducers of MT assembly, omit paclitaxel and adjust the DMSO vol-
umes accordingly.
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