Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

108 Julenius
Fig. 3. A typical sensorgram when the analyte does not associate to the immobilized
ligand. The rise in signal during the association phase is an effect of the bulk
concentration change.
5. Make three samples with the highest concentration (50 nM in the example in
step 3). Each sample should be 150 µL.
6. Run sensorgrams of the three samples, but instead of routinely recording 30 min
of the dissociation phase; record it long enough for the signal to drop significantly,
preferably all the way back to the baseline level. For a strong interaction,
this may take several hours. If the instrument is stable (Note 12) and readily
available, it may in some cases be meaningful to record at least one of the samples
for 24 h or more if the dissociation rate is slow. The dissociation phases of these
sensorgrams will be used to evaluate the dissociation rate constant k off.
7. For evaluation, either start the evaluation software supplied with the instrument,
or export the data into the fitting software of your choice.
8. The dissociation phase must be evaluated first. Delete the first few minutes of the
dissociation phase because they are influenced by the bulk concentration change.
Fit the rest of the dissociation phase to the following equation in the case of a 1:1
complex (see the manual for other stochiometry):
R(t) = C exp(–k off t) + R 0 + R drift t
k off is the dissociation rate constant, t is the time, C is R(t = 0), R 0 is the baseline
value at t = 0, and R drift is the linear slope of the baseline. The drift term (R drift t)
might be excluded if the dissociation rate is fast and the recording time for the
dissociation therefore short (