Biology Reference
In-Depth Information
FIGURE 8.3
A schematic of how MT seeds are bound to the coverslip with each component indicated.
After coating the glass with PPL-PEG biotin, neutravidin (or streptavidin) is added which
reacts with the biotin groups on the surface of the PEG. Finally, the biotin-tubulin-labeled seed
is added which binds to the neutravidin layer.
8.1.4.2
Chemically functionalizing the sample chamber
In order to bind the MT seeds to the
in vitro
sample chamber, we need to chemically
functionalize the glass, for which we use a simple system involving streptavidin-
biotin chemistry. First, the chamber is washed in a solution of PPL-PEG biotin which
coats the surface of the glass very efficiently. To this, a streptavidin layer is added
and finally the biotin-tagged MT seed (see
Fig. 8.3
). Given that the
K
d
of the
streptavidin-biotin interaction is in the order of 10
14
mol l
1
, the binding of
the seeds is extremely efficient.
A simple method of adding components to the sample cell is by flowing in so-
lutions using a pipette and removing themwith a small piece of tissue paper touching
the opposite edge of the coverslip (see
Fig. 8.4
). The capillary action of the tissue
paper draws fluid out of the sample cell. It is important to realize that the sample
cell should never become dry so a balance must be achieved between flowing fluid
in and drawing fluid out. This can take a little practice to get right, and it is most
difficult with the wash steps where larger volumes of buffer are used. Before starting,
make sure that the following is prepared:
