Biology Reference
In-Depth Information
tool that is not available for
in vivo
experiments. For example, by altering the KCl
concentration,
Buey et al. (2011)
noted a change in the binding of GFP-EB3 to the
lattice of MTs, thereby highlighting the electrostatic nature of EB3 binding to MTs
(
Buey et al., 2011
).
Mix the reaction gently with a pipette and transfer to a 5
20-mm Beckman
centrifuge tube. Centrifuge the reaction mix in an airfuge for 10 min at 30 psi, room
temperature. Failure to centrifuge will usually result in the appearance of aggregates
of fluorescent protein, which can be extremely frustrating when collecting data. In
case you do not have access to an airfuge, we have had some limited success
centrifuging the protein mix in a desktop centrifuge but the final results are far from
satisfactory and therefore we highly recommend an airfuge or equivalent centrifuga-
tion prior to protein sample loading. With centrifugation complete, flow the reaction
mix into the sample chamber and finally seal the chamber using vacuum grease. It
might be initially difficult to flow in the sample mix given its higher viscosity, which
is primarily due to the methyl cellulose. If this is the case, make sure that a clean dry
tissue is used and press the tissue firmly to the coverslip (be careful not to break it).
The easiest way to seal the chamber with grease is to take a 2-ml syringe and load a
p200 tip at the base. This makes a small exit for the grease which is loaded into the
syringe. Use this to seal the sample chambers. It is not recommended to use candle
wax as the heat transfer from the molten wax to the sample chamber can be quite high
and this may have deleterious effects on the proteins.
8.1.4.5
TIRF microscopy of the samples
Stable heating for the sample, in our system, is provided by a Tokaihit slide holder and
additionally from the objective heater. If there is only an objective lens heater, then
take note that the sample temperature will be 2-3
C lower than the temperature set
on the objective heater, when the system is at room temperature. We advise to measure
the temperature of the
in vitro
cell with a microthermometer prior to experimentation
to ensure that the sample is at the required temperature (note that the environment, e.g.,
the effects of air conditioning, could potentially alter the temperature). Prior to exper-
iments always allow the TIRF system to warm up to operating temperature. If using a
stage heater, check the manufacturer's instructions but usually it will take around
40 min to reach working temperature. Failure to do this might result in impeded
MT growth (note that the small sample cell size makes it very prone to changes in
the environment). The exact opposite problem, that is, too rapid MT growth and a very
short sample lifetime, can occur in the summer months with a poorly temperature-
regulated TIRF system and environment.
Figure 8.6
shows the results we have
obtained in-house for MTs in the presence of 75 nM GFP-EB3. This image is from
the green channel and hence shows only EB3 interactions, which are clear across
the MT seed, lattice, and plus end where there is an obvious comet. Note that there
is strong binding to the MT seed. This is expected as the GMPCCP should stabilize
the tubulin in a conformation that is a structural mimic of the GTP cap. If using dual
color TIRF, one would easily be able to identify the seeds in the red channel. The back-
ground in this image is fairly standard for the
in vitro
assay. Usually, the background in
the red channel is a little higher. If it is too high, it could be the result of inefficient
