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7.2.2 Kinetic Affinity Experiments
There are some limitations that may make steady state experiments undesirable. For strong
interactions, the amount of analyte required to reach steady state conditions may be excessive.
Also, steady state experiments can be used to determine equilibrium constants K
D
and K
eq
but
not the binding constants k
d
and k
a
. Kinetic affinity experiments can be utilized to determine the
binding constants and equilibrium constants and works effectively for more strongly binding
systems.
Kinetic analysis is done by monitoring binding and dissociation of the analyte to the ligand at
multiple concentrations over time, then applying an interaction model to the data generated
during the experiment. This can be done using TraceDrawer. The figure below illustrates typical
binding curves generated for a kinetic affinity experiment in which 5 different concentrations
were used. Ideally, each concentration should be repeated 3 times. A minimum of 2
concentrations is needed to get reliable kinetic constants. The most typical interaction model
used is a one to one binding model. Unless there is evidence to suggest a more complicated
model, the one to one model should be used.
It is important to limit mass transport effects in kinetic affinity experiments. This can be done by
using a high flow rate for the analyte injection and reducing the surface density of ligand. Also,
data can be fit with models that take into account mass transport to help correct for this effect.
TraceDrawer has this capability. Ideal binding curves are shown below. It is important that
binding curves exhibit curvature at this is how the most accurate theoretical fits will be achieved.
Figure 7.2. Typical binding curves generated from 5 different concentrations of analyte
7.2.3 Recommended Reading
The following are several recommended resources for further reading into binding kinetics:
http://sprpages.nl/
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