Biology Reference
In-Depth Information
cell and 70
L to fill the injection syringe.With fast equilibration times, up to two runs
per hour can be accomplished. Nevertheless, in some cases, a large reaction volume, as
inVP ITC, is still necessary. For example, it might be difficult to obtain or toworkwith
high concentrations of the ligand due to the possibility of aggregation. Also, larger
volumes of reaction might be needed for a low binding constant, or when the
m
H
is
small and requires the sum of many interactions to be detected. In these latter cases,
the ITC200 can still be used, but it will imply conducting several consecutive exper-
iments with the resultant curves concatenated (for a comparison, see
Fig. 18.5
).
D
18.3
RESULTS: TUBULIN/MAPs BY ITC
All the requirements described above may explain why ITC has not been used more
often to study such complex systems as the cytoskeleton network. Nevertheless, ITC
has been used to study the mechanism of bacterial tubulin homologue FtsZ assembly
(
Caplan & Erickson, 2003; Huecas et al., 2007
) or to characterize the binding of sev-
eral modulators of FtsZ assembly in order to use them in new anti-bacterial treat-
ments (
Chen, Milam, & Erickson, 2012; Domadia, Bhunia, Sivaraman, Swarup,
FIGURE 18.5
ITC titration curves (upper panels) and binding isotherms (low panels) of tau-tubulin
interactions registered on ITC200 (left panels) and on VP ITC (right panels) at 10
Cin20mM
NaPi, 0.1 mM GTP, 1 mM TCEP, buffer at pH 6.5. The arrows show syringe refilling with
the same tau solution.
