10
Gives you K
d
, handles a broad range of
molecule types, measures in solution with
very little sample and very low maintenance
Doesn't provide kinetic constants (K
off
and
K
on
) and fluorescent labeling of one of the
binding partners is typically required
environment is altered via any
conformational change or ligand
proximity
6
resulting in a quantifiable,
binding-dependent fluorescence
variation. This variation is measured
and used to calculate the strength
of the molecular interaction or
equilibrium dissociation constant (K
d
) in
an accurate way.
With TRIC it's possible to study the
interactions between a multitude of
molecules (proteins, ions, nucleic acids,
sugars, etc.) and also evalute a very
broad range of interaction strengths
(pico to millimolar). In addition, one
can label the target with fluorophores
that are particularly sensitive to
binding events, resulting in better
signal amplitudes and signal-to-
noise ratios
2
.
Experiments where TRIC
is the major component
of the MST signal
measured were
used in small
molecule or
fragment
screening campaigns as an alternative
to surface-based techniques
17
. TRIC can
quantify interactions in the presence
of organic solvents like DMSO
3
, in cell
lysates
16
or liposomes and nanodiscs
8
.
With measurements taking place in
solution in close-to-native conditions,
the concern of false positives that
occur with NSB of compounds to
immobilization surfaces is eliminated.
Recently it was shown that interactions
can be quantified under conditions
where the measured signal
was derived entirely from
TRIC
14
. The benefit of
this approach is clear
for FBDD — flexible
throughput, fast
time to results,
and a 384-well
plate format
— all desirable
attributes
for screening
campaigns.
