Methods for Studying Microtubule Binding Site Interactions: Zampanolide as a Covalent Binding Agent - Methods in Cell Biology - page 297

g ,4 C, 10 min), collect the 3. Remove aggregates by ultracentrifugation (90,000

supernatant and keep on ice.
4. To measure the protein concentration, dilute a small aliquot 20-fold in 10 mM
phosphate buffer (1% SDS), pH 7.0, and determine the tubulin concentration
from the absorption at 275 nm versus a 20-fold diluted buffer blank. The
extinction coefficient at 275 nm for tubulin is 107,000 M 1 cm 1 ( Andreu,
Gorbunoff, Lee, & Timasheff, 1984 ). Dilute the protein to the required
concentration and alter the buffer if assembly conditions are required.

Tubulin can be assembled into MTs and stabilized by mild glutaraldehyde cross-
linking, and the concentration of taxoid binding sites measured. This protocol is de-
scribed in D ´ az and Buey (2007) . Stabilized MTs are snap-frozen and stored in liquid
nitrogen. Glutaraldehyde-stabilized MTs are an extremely useful tool for studying
MT-ligand interactions and have been essential for identifying the binding site
and binding affinity for a number of important MSAs. Because of the glutaraldehyde
stabilization, these MTs do not assemble or disassemble in response to changes in
ligand concentration, nor are they affected by changes in temperature.

19.1.2 Buffers
Two buffers are required depending on the experimental conditions.

Glycerol-EGTA buffer (GAB)—Assembly buffer
3.4 M glycerol
10 mM sodium phosphate (NaPi)
1 mM EGTA
6 mM MgCl 2
1 mM GTP
pH 6.5

Prepare on the day of use without MgCl 2 and with 0.1 mMGTP, pH 6.8, keep on ice.
After column equilibration, add 6 mM MgCl 2 , and increase the GTP to 1 mM for
assays requiring assembly conditions (pH shifts to 6.5). In experiments that use
stabilized-MTs, the GAB buffer left over from the dialysis is used to dilute the sites
and act as the experimental buffer.

Phosphate-EDTA buffer (PEDTA), 0.1 mM GTP—Buffer unfavorable for
assembly
10 mM NaPi
1 mM EDTA
0.1 mM GTP
pH 7.0

Prepare on the day of use, keep cold on ice. When required, increase the GTP con-
centration to 1 mM and add the required amount of MgCl 2 .