Biology Reference
In-Depth Information
FIGURE 8.4
How to flow components into the sample chamber. The double-sided tape, shown in light gray,
is cut so as to have a little extra on the inflow side of the sample chamber. When adding
buffer, this makes the process easier by directing the solution toward the coverslip and inhibits
buffer from spreading out across the glass when trying to flow it through the sample chamber.
MRB80 buffer both on ice and warmed to 37 C.
Streptavidin stock diluted by adding 40
l of MRB80. Keep at 37 C.
m
One
-casein stock on ice.
An additional
k
-casein stock, which is diluted by adding 40
l of MRB80 and
k
m
kept at 37 C.
All other components for the assay are kept on ice.
Another useful addition is some form of humidity chamber so that the sample cell
does not dry out. In our lab, we make use of standard 10 cm circular cell culture
dishes to which we add a small piece of water-soaked tissue paper. The sample cell
is left inside this dish, on a makeshift platform, with the lid closed during incubation
steps so as to prevent drying out of the sample chamber. All of the following steps can
be carried out at room temperature. With everything prepared and assuming a 10-
l
m
sample cell, carry out the following to prepare the sample chamber:
Flow in 10
l of PPL-PEG biotin. Allow binding for 5 min.
To remove unbound PEG biotin, flow through 200
m
l of warm MRB80 buffer.
m
Flow through 20
l of the diluted prewarmed streptavidin. Incubate for 3 min.
To remove excess streptavidin, flow through 50
m
l of warm MRB80 buffer.
With these steps complete, the sample chamber is now functional and ready to
bind the MT seeds.
m
8.1.4.3 Attaching the seeds and blocking the chamber
While the streptavidin is incubating, retrieve the GMPCCP-stabilized MT seeds from
the 80 C freezer. Under no circumstances place them on ice or they will depoly-
merize. After removing the aliquot from the freezer, it is best to keep it warm in your
hand until placing it in a 37 C heating block. Given the small volume of the seed
stock, it is best to use a heating block with a heated lid so as to avoid evaporation.
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