monitor the reaction on shorter and longer timescales to look
for additional kinetic phases (see Note 10). It is advisable to
collect at least four individual recordings and average them
before proceeding to analysis. If the signal-to-noise is poor or
the model ambiguous then more acquisitions can be made and
averaged. Sometimes one or more trace(s) in a series of acquisitions displays obvious artifacts. Such traces should be discarded
and not included in the average. However, to reduce any risk of
bias by “cherry picking,” any trace that is removed from the
data set should be replaced with a couple of new acquisitions.
9. If the experiment gives a clear kinetic trace, it should be fitted
to a single exponential equation with a fixed end point, preferably using the software provided with the instrument since it
provides instant feedback for how to proceed. Initial data
points should be removed according to the mixing time analysis (see Note 1). The residuals of the curve fitting should be
inspected. Are these residuals randomly distributed over the
entire recording or are there systematic deviations? In the event
of nonrandom residuals, a second exponential term should be
included and the data refitted. In rare cases, triple exponential
transients are observed. Judging if a trace is appropriately fit or
whether additional exponentials are needed is a critical part of
kinetic studies. Figure 1 provides examples of fitting to three
sets of experimental data.
10. After observing initial kinetic traces it may be useful to reconsider the concentration of protein A. The recommendation of
1 μM of A is a good starting point but if there is sufficient signal
amplitude then this concentration can be reduced. The greater
the ratio of [B]/[A] the more optimal are pseudo-first-order
conditions. On the other hand, sometimes a higher concentration of A may be required to obtain a clear change in fluorescent signal upon binding, in particular, if intrinsic Tyr
fluorescence is monitored.
11. The next step, whether a single, double, or triple exponential is
observed, is to measure binding at different concentrations of
B while keeping the concentration of A constant and check
how k obs changes. Observe if any systematic deviations in the
fitting residuals are reproducible and present at all concentrations of B.
Having collected data at different concentrations of B and fit
these to an appropriate model allows for some inferences about
binding mechanism as is explained and exemplified in more detail
using a number of case studies below.
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Elin Karlsson and Per Jemth
for additional kinetic phases (see Note 10). It is advisable to
collect at least four individual recordings and average them
before proceeding to analysis. If the signal-to-noise is poor or
the model ambiguous then more acquisitions can be made and
averaged. Sometimes one or more trace(s) in a series of acquisitions displays obvious artifacts. Such traces should be discarded
and not included in the average. However, to reduce any risk of
bias by “cherry picking,” any trace that is removed from the
data set should be replaced with a couple of new acquisitions.
9. If the experiment gives a clear kinetic trace, it should be fitted
to a single exponential equation with a fixed end point, preferably using the software provided with the instrument since it
provides instant feedback for how to proceed. Initial data
points should be removed according to the mixing time analysis (see Note 1). The residuals of the curve fitting should be
inspected. Are these residuals randomly distributed over the
entire recording or are there systematic deviations? In the event
of nonrandom residuals, a second exponential term should be
included and the data refitted. In rare cases, triple exponential
transients are observed. Judging if a trace is appropriately fit or
whether additional exponentials are needed is a critical part of
kinetic studies. Figure 1 provides examples of fitting to three
sets of experimental data.
10. After observing initial kinetic traces it may be useful to reconsider the concentration of protein A. The recommendation of
1 μM of A is a good starting point but if there is sufficient signal
amplitude then this concentration can be reduced. The greater
the ratio of [B]/[A] the more optimal are pseudo-first-order
conditions. On the other hand, sometimes a higher concentration of A may be required to obtain a clear change in fluorescent signal upon binding, in particular, if intrinsic Tyr
fluorescence is monitored.
11. The next step, whether a single, double, or triple exponential is
observed, is to measure binding at different concentrations of
B while keeping the concentration of A constant and check
how k obs changes. Observe if any systematic deviations in the
fitting residuals are reproducible and present at all concentrations of B.
Having collected data at different concentrations of B and fit
these to an appropriate model allows for some inferences about
binding mechanism as is explained and exemplified in more detail
using a number of case studies below.
114
Elin Karlsson and Per Jemth