9 Kinetics and affinity analysis
9.4 Quality assessment for kinetics evaluation
166
Biacore T100 Software Handbook BR-1006-48 Edition AE
Bulk refractive index
After reference subtraction and blank subtraction, sensorgrams for kinetic
evaluation should not in principle contain any bulk refractive index shifts
(parameter
RI
in predefined models). However, there may be some
circumstances where small bulk refractive index shifts may remain in reference-
and blank-subtracted data. On the other hand, the fitting algorithm tends to
interpret rapid interaction events (incorrectly) as bulk shifts. If the fitting returns
significant values for
RI
, a warning will be issued in the quality control tab.
Examine the sensorgrams and fitted curves to determine whether bulk shifts as
reported by the fitting are true or false. In cases where reported bulk shifts are
unreasonably large, you may want to set
RI
to a constant value of zero in the
Parameters
setting for the fitting. If you do this, the bulk contributions
component in the quality control tab will be reported as neutral since the
RI
parameter was not evaluated.
Sensorgram curvature
You should check that the sensorgrams have sufficient curvature for kinetic
determination. Ideally, the sensorgrams for at least the one or two highest
concentrations should show measurable binding rates at the beginning of the
sample injection and approach a steady state towards the end of the injection.
Sensorgrams that approximate to “square-wave” pulses (indicating rapid
association and dissociation) and those that do not flatten out during the
injection generally do not contain sufficient kinetic information for reliable
evaluation. Ideally, the dissociation phase should be long enough to monitor a
fall in response of at least 10-15% of the starting value.
Figure 9-2.
Examples of sufficient and insufficient sensorgram curvature.
Top: Ideal sensorgrams approaching steady state during sample injection and returning
to baseline during dissociation.
Bottom left: Rapid interaction approaching “square wave” appearance. These
sensorgrams return rate constants close to the limit of measurement for the instrument.
Bottom right: Slow association and dissociation, giving insufficient curvature in both
association and dissociation phases. Evaluation is possible but will not be very reliable.
Summary of Contents for Biacore T100
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