Biology Reference
In-Depth Information
interaction. Fortunately, modern microcalorimeters allow carrying out titration ex-
periments at a wide range of temperatures.
18.2
TUBULIN AND MAPs SAMPLE PREPARATION
Because of certain peculiarities of tubulin, its interaction with regulatory proteins has
been studied by ITC only occasionally, despite the growing popularity of this method
and its obvious advantages. The difficulties that could arise during such a study ne-
cessitate a deep knowledge of both method details and tubulin cytoskeleton regula-
tory mechanisms. In this section, we want to draw attention to some important points
about tubulin and certain MAPs sample preparations for ITC experiments.
18.2.1
Equilibration
To minimize the heat signal due to the dilution of the samples during injections, a
balance between the composition of buffers in the calorimetric cell and the syringe
needs to be established. Due to the high sensitivity of microcalorimeters, the two so-
lutions must be matched with regard to composition, pH, buffer, and salt concentra-
tions. A slight mismatch between the two solutions may lead to heat of dilution that
could overwhelm the heat of the binding reaction. Usually, to achieve the perfect
match between buffers in the cell and syringe, dialysis of both interactant solutions
against the same buffer is used. Unfortunately, due to tubulin instability, buffer spec-
ificity, and the necessity of keeping a high concentration of ligand, this option is not
appropriate. After purification, when tubulin is stored, 1 M sucrose buffer to stabilize
its conformation upon freezing (
Frigon & Lee, 1972
), the sucrose should be
completely removed from buffer before ITC experiment, since it significantly con-
tributes to the dilution effect. However, extensive dialysis cannot be used because of
low tubulin stability over an extended period of time in the absence of a stabilizer.
Previously, we described a tubulin equilibration procedure using two custom-made
columns filled with Sephadex G25 (
Andreu & Timasheff, 1982; Barbier, Peyrot,
Leynadier, & Andreu, 1998; Devred et al., 2010; Na & Timasheff, 1982; Peyrot
et al., 1992
). Later, we optimized the protocol by replacing these two columns by
a single desalting Hitrap column (GE Healthcare) on an AKta Purifier FPLC system.
This allowed us to reduce the time and to increase the yield of tubulin preparation.
Tubulin can also be commercially bought as a powder, which contains stabilizers of
tubulin that should be removed by running the tubulin preparation on a desalting col-
umn. MAPs, such as stathmin and tau, can be dry-lyophilized and then stored as pow-
ders (
Devred et al., 2004, 2008
). Direct dilution of lyophilized proteins in
experimental buffer often results in an increase in the dilution signal, even if MAPs
were dialyzed against water to eliminate salt before dry-lyophilization. Thus, when
used for ITC experiments, dry-lyophylized MAPs should be resuspended in the
buffer of interest, centrifuged to remove aggregated proteins, and ran on the desalting
column identical to the one used for tubulin.
