Purification and Biophysical Analysis of Microtubule-Severing Enzymes In Vitro - Methods in Cell Biology - page 198

A Top view of chamber

B Side view of flow chamber

Glass slide

Glass coverslip

Flow sample
here

C Zoom in of flow chamber

Double-stick
tape

Severing enzyme

Microtubule

TIRF evanescent wave

Pluronic F-127
Antibody

Laser illumination

FIGURE 13.3
Flow chamber diagram. (A) A diagram of the top view of flow chamber. The coverslip is
closest to the objective. It is stuck to the glass coverslip with double-stick tape to make a
chamber you can flow into. (B) A side view of the chamber. (C) A zoomed-in view of the
chamber showing the surface preparation. Antitubulin antibody (yellow) is bound and
Pluronic F-127 (orange) is added to block the glass. Microtubules are added to the
chamber to bind to the tubulin antibodies. Finally, a solution of severing enzymes, ATP,
glucose, DTT, and an oxygen scavenging system are flowed into the chamber. The severing
enzymes are depicted by the green hexamers on the microtubule. We use the evanescent
field of the TIRF illumination to visualize the severing enzymes.

avoid any leakage, gently press the coverslip that is in contact with the
double-sided tape.
2. A 1:100 dilution of monoclonal anti-alpha tubulin antibody (2%, v/v) is prepared
in PEM-100 and flowed in to fill the chamber.
3. The antibody is incubated at room temperature for 5 min.
4. Flow 5% Pluronic F-127 prepared in PEM-100. Centrifuge the F-127 solution
prior to use each day. Use a filter paper to absorb the liquid from the other
end to create flow. The chamber is again incubated at room temperature for
5 min to bind F-127 to open hydrophobic surfaces.
5. Flow in a 1:100 dilution of microtubules. See previous section on microtubules.
Incubate the chamber at room temperature for 5 min.
6. To remove any nonattached microtubules, flow in a chamber wash buffer
made of PEM-100 with or without 40 m M Taxol depending on the type of
microtubules used. At this point, the chamber is ready to be used.

13.1.3 Biophysical assays
We have published results using several biophysical assays to study the activity of
katanin severing (Diaz-Valencia et al., 2011; Mukherjee, 2012; Zhang, 2011). In all
cases, we use fluorescence imaging to examine severing of microtubules. Here, we
describe how to perform these experiments in detail.