Biology Reference
In-Depth Information
If this is the first use of the seed stock, then it will be necessary to check MT integrity.
It is also important to test the best seed dilution for
in vitro
experiments. With our
TIRF setup, we use a QuantEM 512SC camera (Roper Scientific) with a 512
512
pixel area, 0.065
m per pixel. We aim to have a seed dilution that gives approxi-
mately 3-5 seeds in this field of view. Usually, with a standard seed stock, this is
a 1:130 dilution, but depending upon the seed preparation the dilution can vary from
1:100 to 1:160. It is usually best to prepare a few sample chambers to test the best
seed dilution prior to experiments. Once the dilution has been obtained, it can be used
for the entire stock of frozen seeds, so it is worth investing a little time to be precise.
For a TIRF image of MT seeds, see
Fig. 8.5
.
With the sample chamber prepared, take 1
m
l of seeds and dilute as appropriate in
m
warm MRB80 buffer. Flow in 50
l of the diluted seed stock into the streptavidin-
prepared sample chamber. Throw the remaining dilution away. Once the seeds have
been diluted, they become unstable so do not use a diluted seed solution for a next
round of experiments (it is of course fine to use one seed dilution if carrying out a
number of experiments in parallel). Incubate the reaction for 5 min and make sure
that the sample chamber is at room temperature (see
Section 1.4.4
). After 5 min,
wash the chamber by flowing in 50
m
m
l of warm MRB80. Finally, flow in 30
m
lof
warm diluted
-casein (this acts as a blocking agent to inhibit further binding to
the sample chamber surface) and move on to the preparation of the
in vitro
sample.
k
FIGURE 8.5
A TIRF image of GMPCCP-stabilized microtubule seeds labeled with X-Rhodamine tubulin.
The seed density in this image would be considered too high but is shown for demonstration
purposes. Ideally, one should aim for 3-5 seeds per field of view. The dappled effect in the
seeds is due to bleaching of the X-Rhodamine during imaging. The imaging conditions
employed were 3 mW 561 nm laser, 100
objective, 500 ms exposure time.
