SPAR2021-ET
The ability to determine the binding affinity of lipids to proteins is
an
essential part of understanding protein-lipid interactions in membrane
trafficking, signal transduction and cytoskeletal remodeling. Classic
tools for
measuring such interactions include surface plasmon resonance (SPR)
and
isothermal calorimetry (ITC). While powerful tools, these approaches
have
setbacks. ITC requires large amounts of purified protein as well as
lipids, which
can be costly and difficult to produce. Furthermore, ITC as well as
SPR are very
time consuming, which could add significantly to the cost of
performing these
experiments. One way to bypass these restrictions is to use the
relatively new
technique of microscale thermophoresis (MST). MST is fast and cost
effective
using small amounts of sample to obtain a saturation curve for a given
binding
event. There currently are two types of MST systems available. One
type of MST
requires labeling with a fluorophore in the blue or red spectrum. The
second
system relies on the intrinsic fluorescence of aromatic amino acids in
the UV
range. Both systems detect the movement of molecules in response to
localized
induction of heat from an infrared laser. Each approach has its
advantages and
disadvantages. Label-free MST can use untagged native proteins;
however, many
analytes, including pharmaceuticals, fluoresce in the UV range, which
can
interfere with determination of accurate KD values. In comparison,
labeled MST
allows for a greater diversity of measurable pairwise interactions
utilizing
fluorescently labeled probes attached to ligands with measurable
absorbances in
the visible range as opposed to UV, limiting the potential for
interfering
signals from analytes.
