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Figure 1.
Fig. 1. Measuring the affinity and kinetics of DAF[234]
binding to EV11 by surface plasmon resonance. A, DAF[234] (12
µM) was injected (bar) for 22.5 s over sensor surfaces
with a high level of (3020 RU), a low level of (1370 RU), or no
(control) EV11 immobilized. An overlay plot of all three
sensorgrams is shown, normalized to the same preinjection level.
Inset, same sensorgrams after subtraction of the control
sensorgram. In B, DAF[234] was injected (bar) at the indicated
concentration for 22.5 s over sensor surfaces with EV11 (3020
RU) or no protein immobilized. An overlay plot is shown of all
five sensorgrams after subtraction of their respective control
sensorgrams. Curves, a fit of the simple Langmuir binding model
(A + B  AB) to all
sensorgrams simultaneously gives a k[on] of 140,000 M  1·s
1 and a
k[off] of 0.3 s 1. The
curve fitting was performed by numerical integration using the
global analysis option (i.e. simultaneous fitting of the
association and dissociation phases) of BIAevaluation 3.0
(BIAcore AB). C, plot of the equilibrium response obtained in A
during injection of different DAF[234] concentrations. Nonlinear
curve fitting of the Langmuir binding model to these data (bar)
gives a K[D] of 2.8 µM. Inset, Scatchard plot of the same
data. A linear fit of these data (bar) also gives a K[D] of 2.8
µM.
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