Biology Reference
In-Depth Information
8.1.4.4
Preparation of the protein samples
Now that the sample chambers have been prepared, it is time to make the primary
protein mix. It is here that we will add not only the components required for efficient
MT growth but also the specific MT-binding proteins under study. For a good work
flow, it is recommended to make this mix after the wash step prior to seed attach-
ment. Add the following in a microtube:
6.5
l MRB80 buffer.
m
2.0
l of 1% methyl cellulose.
m
3.0
l of 333 mM KCl (50 mM final).
m
lof5mgml
1
2.0
-casein.
m
k
0.5
l of 50 mM GTP (1.2 mM final).
m
3.0
lof100
M tubulin (15
M final).
m
m
m
2.0
l
þ
TIP (
75 nM final concentration for ensemble and
0.6 nM for single-
m
molecule experiments).
0.5
l oxygen scavenger mix.
m
0.5
l of 1 M glucose.
m
The concentration of 75 nM final for the
TIP is a good starting point, as with mam-
malian EB proteins this concentration is known to produce “comets.” However, the
final concentration used will ultimately depend on the
þ
þ
TIP under study. For
single-molecule experiments, we have listed 0.6 nM as a good starting point based
on EB3 studies (
Montenegro Gouveia et al., 2010
). This will provide good coverage
of the GDP lattice and plus-end interactions at a density appropriate for analysis. If
carrying out a single-molecule experiment, it is a good idea to have some form of tracer
to visualize growingMTs as the protein under study will be at too small a concentration
to see the MT (end). Previous studies have used a higher concentration (75 nM) of the
same protein that is studied in single-molecule “mode,” using different fluorescent pro-
teins (and hence channels) to distinguish low and high concentrations (
Montenegro
Gouveia et al., 2010
). This allows one to easily identify the MT in one channel and
position single molecules in the other. An alternative method is to label the tubulin
instead. This can be done by adding a small
tracer amount of, for example,
X-Rhodamine or Alexa tubulin (
0.13
M). The MTs will be faintly visible in the
m
red channel and the
TIP can then be identified in the green channel. Sometimes it
is advisable to optimize the concentration of tubulin tracer as too high a concentration
will lower the signal-to-noise ratio in the TIRF resulting in a “white out.”
Although GFP is widely used, it is not the best choice for single-molecule exper-
iments, primarily due to its blinking behavior. We have seen blinking of GFP in the
20- to 30-ms time scale, which has impeded fast time exposure imaging (e.g., 10 ms)
of EB proteins. One way to overcome this is to use alternate fluorophores such as
Alexa dyes. However, the preparation of protein samples becomes more complex
with such methods.
The components and their concentrations listed above are considered the standard
setup for the
in vitro
assay. However, it should be obvious that the possibility to alter
a given component and study its subsequent effect provides a unique and powerful
þ
