ligand in Tube 1 must be identical to that pipetted into Tubes
2–16.
20. Strong trends in pre-IR fluorescence could be due to several
phenomena. The most common is a fluorescence increase with
ligand concentration, potentially indicating that the ligand has
passivated sticky plasticware and thus there is more of it to
detect in the high-ligand-concentration samples. The manufacturer recommends a control experiment in which mixtures
with the highest and lowest ligand concentrations are centrifuged in a benchtop microcentrifuge at its maximum speed for
10 min, followed by the addition of equal volumes of a solution
containing 4% (w/v) sodium dodecyl sulfate and 40 mM
dithiothreitol. After incubation of these solutions at 95
C for
10 min, they are placed into capillary tubes and a capillary scan
is performed; if the difference has disappeared, this suggests
that the interaction of protein and ligand was responsible for
the difference and thus it is valid to use the flourescence signal
for the analysis.
21. Data with a strong signal (e.g., b
F ! 10 ; see Eq. 6) in the
differential bleaching of the fluorophore should not be analyzed for using T-Jump or thermophoresis. Instead, the differential bleach signal can be analyzed if it can be observed
reproducibly [13].
22. For this analysis, Tube 10 appeared to be an outlier, and data
derived from this sample were excluded from these sample
analyses. A binding-curve data point can be excluded by leftclicking on it in PALMIST.
23. Differences in opinion exist among MST practitioners
concerning which MST power to use; some would see the
20% MST-power data in Table 1 and find this to be adequate.
Their notion is that the lowest MST power possible should be
used to minimally perturb the system. However, in systems in
which T-Jump will be used for analysis, we almost always use
the MST power that results in the highest b
F , while confirming
that the MST traces are smooth, i.e., have no signs of aggregation. Because T-Jump analyzes data just after actuation of the
IR laser, the effects of the high ΔT are not as deleterious as they
are to thermophoresis.
24. By averaging the data, statistics on the standard deviation of F n
at each concentration are obtained. These can be used as
weights in the fitting by clicking on “Use Weighted Fitting”
in the panel at the right side of PALMIST. However, we do not
recommend doing so; Monte Carlo simulations indicate that
fitting outcomes are slightly worse when using such a fitting
scheme (C.A.B., unpublished results).
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Shih-Chia Tso and Chad A. Brautigam
2–16.
20. Strong trends in pre-IR fluorescence could be due to several
phenomena. The most common is a fluorescence increase with
ligand concentration, potentially indicating that the ligand has
passivated sticky plasticware and thus there is more of it to
detect in the high-ligand-concentration samples. The manufacturer recommends a control experiment in which mixtures
with the highest and lowest ligand concentrations are centrifuged in a benchtop microcentrifuge at its maximum speed for
10 min, followed by the addition of equal volumes of a solution
containing 4% (w/v) sodium dodecyl sulfate and 40 mM
dithiothreitol. After incubation of these solutions at 95
C for
10 min, they are placed into capillary tubes and a capillary scan
is performed; if the difference has disappeared, this suggests
that the interaction of protein and ligand was responsible for
the difference and thus it is valid to use the flourescence signal
for the analysis.
21. Data with a strong signal (e.g., b
F ! 10 ; see Eq. 6) in the
differential bleaching of the fluorophore should not be analyzed for using T-Jump or thermophoresis. Instead, the differential bleach signal can be analyzed if it can be observed
reproducibly [13].
22. For this analysis, Tube 10 appeared to be an outlier, and data
derived from this sample were excluded from these sample
analyses. A binding-curve data point can be excluded by leftclicking on it in PALMIST.
23. Differences in opinion exist among MST practitioners
concerning which MST power to use; some would see the
20% MST-power data in Table 1 and find this to be adequate.
Their notion is that the lowest MST power possible should be
used to minimally perturb the system. However, in systems in
which T-Jump will be used for analysis, we almost always use
the MST power that results in the highest b
F , while confirming
that the MST traces are smooth, i.e., have no signs of aggregation. Because T-Jump analyzes data just after actuation of the
IR laser, the effects of the high ΔT are not as deleterious as they
are to thermophoresis.
24. By averaging the data, statistics on the standard deviation of F n
at each concentration are obtained. These can be used as
weights in the fitting by clicking on “Use Weighted Fitting”
in the panel at the right side of PALMIST. However, we do not
recommend doing so; Monte Carlo simulations indicate that
fitting outcomes are slightly worse when using such a fitting
scheme (C.A.B., unpublished results).
180
Shih-Chia Tso and Chad A. Brautigam