Table of Contents
Advertisement
where [A] is the concentration of free fluorescent molecule, [L] the concentration of free ligand and
[AL] is the concentration of the complex of A and L. The free concentrations of A and L are [A]=[A
]-
0
[AL] and [L]=[L
]-[AL], respectively. [A
] is the known concentration of the fluorescent molecule and
0
0
[L
] is the known concentration of added ligand. This leads to a quadratic fitting function for [AL]:
0
2
1/2
[AL]=1/2*(([A
]+[L
]+ K
)-(([A
]+[L
]+ K
)
-4*[A
]*[L
])
)
0
0
d
0
0
d
0
0
The concentration of fluorescent molecule [A
] is kept constant during the experiments and the
0
concentration of ligand [L
] is varied in a dilution series. The signal obtained in the measurement
0
directly corresponds to the fraction of fluorescent molecules that formed the complex x=[AL]/[A
],
0
which can be easily fitted with the derived equation to obtain K
.
d
Figure 1: MST setup and experiments. (A) Schematic representation of MST optics. MST is measured in capillaries with
a total volume of down to 4 µl. The fluorescence within the capillary is excited and detected through the same objective. A
focused IR laser is used to locally heat a defined sample volume. The MST response of fluorescent molecules within the
temperature gradient are detected. (B) Typical signal of an MST trace. Initially, the molecules are homogeneously
distributed and a constant ''initial fluorescence'' is detected. After activation of the IR laser, a drop in fluorescence is
observed, which corresponds to TRIC triggered by the fast temperature change and thermophoretic movement of the
fluorescent molecules out of the heated sample volume. After deactivation of the IR laser, a fluorescence increase occurs,
caused by the 'backdiffusion' of molecules and a decrease in sample temperature. MST, MicroScale Thermophoresis; IR,
infrared. (C) Typical binding experiment. The MST signal of a fluorescent molecule (black trace; ''unbound'') changes upon
binding to a non-fluorescent ligand (grey trace, intermediate state; red trace, ''bound''), resulting in different traces. (D) For
analysis, the change in MST signal is expressed as the change in the normalized fluorescence (∆F
), which is defined
norm
. Titration of the non-fluorescent ligand results in a gradual change in MST, which is plotted as ∆F
as F
/F
against the
1
0
norm
ligand concentration to yield a dose-response curve, which can be fitted to derive binding constants.
16
Hide quick links:
Advertisement
Table of Contents
Need help?
Do you have a question about the Monolith NT.115 and is the answer not in the manual?
Questions and answers