Surface Plasmon Resonance can be used for both kinetic and equilibrium analyses. This post will provide background on each type of experiment and explain what information you can expect to obtain from each.
The main advantage doing a kinetic analysis is that you can obtain the association rate constant (ka), the dissociation rate constant (kd), and the equilibrium dissociation constant (KD) all from one set of experiments. KD is a measure of two molecules’ affinity for each other (how strong the interaction is between the two) and ka and kd tell how quickly they associate (on rate) and dissociate (off rate), respectfully.
In order to achieve the best results from your kinetic analysis, it is very important to have curvature in the association part of the curve) and at least a 10% decrease in response for the dissociation (see the picture below). You should also inject five or more sample concentrations at least in duplicate to show stability and improve accuracy.
A disadvantage of doing kinetic analysis can be that you consume a considerable amount of sample with flow rates of 25-35 µL/min and injection times of 3-10 minutes for slower associations (fast associations usually only need to be run for 1-2 minutes).
A typical kinetic sensorgram looks like this:
A disadvantage of equilibrium analysis can be that information obtained from equilibrium analysis is more limited since only KD is calculated. But, an advantage can be that sample consumption is relatively low - flow rates of 5-10 µL/min for a 100 µL injection can be used.
Results from equilibrium analysis are plotted in the form of a Langmuir binding isotherm to determine a KD value. A Langmuir binding isotherm can be obtained by plotting the maximum response from a series of sample injections of different concentrations versus concentration. When choosing the maximum response, use the same time (point or interval) near the end of the association.
It is important to allow association times to be long enough to achieve saturation at most concentrations. This amount of time can vary greatly based on the analyte’s affinity for the target and the target density.
The following is an example of an experiment where equilibrium (the binding isotherm) and kinetic data (inset) can be obtained from the same experiment. KD values agree for both experiments: KD = 809nM from the kinetic fit and KD = 940nM from the Langmuir isotherm.
Keep in mind that even though you can try to do both kinetic and equilibrium fits from one set of experiments, the values obtained from the kinetic fit and equilibrium fit only agree if you can achieve saturation in your experiment.
We hope you enjoy this column and return regularly for future posts, which will provide additional tips for when you perform use Reichert systems to perform Surface Plasmon Resonance. You can read more about SPR elsewhere on Reichert’s website, including our first blog post and an earlier blog post on the importance of rate constants.
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