Biology Reference
In-Depth Information
and buffer plus GTP at twice its desired final concentration. The two halves are then
combined. The steps in this procedure are the following:
1.
Prewarm the spectrophotometer to 30
C (setting this will depend on the
instrument—with the SpectraMax Plus, it is simply entered into the software setup).
2.
Prepare the sample in a small microcentrifuge tube on ice. The sample consists of
30
l of the tubulin solution at 2.5 mg/ml (twice the desired final concentration)
in PM buffer with 1.0 MTMAO. If a drug is to be added, it can be added in 3
m
lof
DMSO to the tubulin solution and preincubated in a small microcentrifuge tube
on ice. Control reaction would have 3
m
m
l of DMSO alone.
3.
The final 27
m
l of solution consisting of PMbuffer with 1.0 MTMAO, 1 mMGTP.
This ismixedbypipettingupanddown, and the total transferred to the cuvette. In this
sort of experiment with multiple samples, we would prepare a larger volume of the
PM-TMAO-GTP solution and just add 27
m
l of the mixture per sample.
4.
After adding the sample to the cuvette (being careful to avoid air bubbles), place
it in the spectrophotometer and record OD (350 nm) versus time. A total time of
20 min and a 20-s interval between readings is usually sufficient.
5.
(Optional). At the end of the recording period, the sample is removed with a
micropipet and centrifuged for 5 min at full speed in an Airfuge (
200,000
g
),
or 15 min at full speed (
20,000
g
) in a room-temperature microcentrifuge.
The top 30
m
l is removed and used for protein assay of the supernatant, in order to
determine
e
* (see
14.3.4
Analysis of Turbidity).
14.3.3
Turbidity assay in multiwell plates
The sample preparation is very similar to that in the cuvette assay, but modified for
multiwell plates.
1.
Set the reading chamber of the plate reader to 30
C.
2.
Prepare two small rectangular styrofoam boxes with openings sufficient to hold
a multiwell plate. Fill one with ice and the other with
1-2 cm of room-
temperature water.
3.
Place the half-area, 96-well plate on the ice.
4.
Place test compounds in 3
m
l DMSO into each well. Also perform the following
controls: 3
l of DMSO alone, to assess the effect of the vehicle DMSO on the
polymerization, and 3
m
l of 1 mM podophyllotoxin as a negative (no
polymerization) control.
5.
Pipet the tubulin solution (30
m
m
l, 2.5 mg/ml) into each well and mixed by
stirring with the pipet tip.
6.
Keep the plate on ice until all wells have samples, and longer if a preincubation
with test compounds is desired.
7.
Carefully add 27
m
l of the PM-GTP-TMAO solution carefully to each well and
mix the sample by up and down pipetting, being careful to avoid bubbles (this
takes practice).
8.
Move the plate to the styrofoam water box and float it on the water for 1 min.
Timing this step carefully will make the experiment quite reproducible.
