(NBS). The mixing time measurement should be performed
under the same conditions of buffer and mixing ratio (1:1,
1:10, etc.) as used in the binding experiment. The reaction
kinetics between NATA (0.5–1 μM) and an excess NBS are
measured over a range of 5–6 different NBS concentrations
that depends on the temperature of measurement but usually in
the range of 100–2000 μM. The k obs values measured should
be between 200 and 1000 s
À1 . The entire set of kinetic traces
are then fit simultaneously to single exponential functions.
Then, some early data points are removed systematically across
the data set and the fit repeated iteratively until all the exponentials intersect at a common “time zero.” It is very difficult
to obtain data with sufficient quality that all traces indeed will
intersect in a single point. But it is usually easy to determine a
time region where all traces are close to each other. This is the
true time zero with regard to the mixing of NATA and NBS
under the experimental conditions used. The mixing time is the
time from time zero to the time of the first good data point,
i.e., where data were excluded/included to obtain the curve fit
giving a good enough intersection of all single exponentials
(Fig. 5).
0.00
0.05
0.10
-0.2
0.0
0.2
-0.002 -0.001 0.000 0.001 0.002 0.003 0.004
4
6
8
10
0.00
0.02
0.04
0.06
0.08
0.10
4
6
8
10
a
b
Time (s)
Fluorescence intensity
Fluorescence intensity
Residuals
Time (s)
Time (s)
≈3 ms
Fig. 5 Determination of mixing (dead) time in a stopped flow. (a) The reaction between NATA (1 μM) and NBS
(500–900 μM) was measured by excitation at 280 nm and using a 320 nm long-pass emission filter. Each
trace was fitted to a single exponential. (b) When experimental data points up to 2 ms were removed, the fitted
curves intersected around À1 ms, which is the true time zero for the measured reaction. The first data points,
which fall on the fitted curve are around 2 ms. Thus, the mixing time, or dead time, is 3 ms, which is the time
between time zero and complete mixing
Kinetics of IDP Binding
127
under the same conditions of buffer and mixing ratio (1:1,
1:10, etc.) as used in the binding experiment. The reaction
kinetics between NATA (0.5–1 μM) and an excess NBS are
measured over a range of 5–6 different NBS concentrations
that depends on the temperature of measurement but usually in
the range of 100–2000 μM. The k obs values measured should
be between 200 and 1000 s
À1 . The entire set of kinetic traces
are then fit simultaneously to single exponential functions.
Then, some early data points are removed systematically across
the data set and the fit repeated iteratively until all the exponentials intersect at a common “time zero.” It is very difficult
to obtain data with sufficient quality that all traces indeed will
intersect in a single point. But it is usually easy to determine a
time region where all traces are close to each other. This is the
true time zero with regard to the mixing of NATA and NBS
under the experimental conditions used. The mixing time is the
time from time zero to the time of the first good data point,
i.e., where data were excluded/included to obtain the curve fit
giving a good enough intersection of all single exponentials
(Fig. 5).
0.00
0.05
0.10
-0.2
0.0
0.2
-0.002 -0.001 0.000 0.001 0.002 0.003 0.004
4
6
8
10
0.00
0.02
0.04
0.06
0.08
0.10
4
6
8
10
a
b
Time (s)
Fluorescence intensity
Fluorescence intensity
Residuals
Time (s)
Time (s)
≈3 ms
Fig. 5 Determination of mixing (dead) time in a stopped flow. (a) The reaction between NATA (1 μM) and NBS
(500–900 μM) was measured by excitation at 280 nm and using a 320 nm long-pass emission filter. Each
trace was fitted to a single exponential. (b) When experimental data points up to 2 ms were removed, the fitted
curves intersected around À1 ms, which is the true time zero for the measured reaction. The first data points,
which fall on the fitted curve are around 2 ms. Thus, the mixing time, or dead time, is 3 ms, which is the time
between time zero and complete mixing
Kinetics of IDP Binding
127