More things to think about related to Surface Plasmon Resonance data analysis:

1) To determine an accurate dissociation rate constant (kd), you need to observe at least a 10% decrease in the dissociation signal. For biomolecular interactions with slow dissociation rates, run at least one of your sample dissociations for a longer time to obtain the kd, and use the value obtained from that injection when globally fitting your sensorgrams. Alternatively you can program all of your sample injections to have longer dissociation times if time is not an issue in your experiments.



2) If you are unsure what model your data should fit to, try globally fitting your responses to a 1:1 binding model to obtain an estimate of the equilibrium dissociation constant (KD). If the 1:1 model (one phase exponential association and decay) does not adequately fit the shape of the association and/or dissociation responses, then you can try other models. In the example below, the dissociation portion of the curves fit poorly when using a 1:1 binding model, but both ka and kd fit well using a 1:2 binding model.


Data fit to 1:1 binding model – dissociations are not single exponential.



Data fit to 1:2 binding model - curves fit well to this model.

3) You also can use Surface Plasmon Resonance (SPR) to obtain thermodynamic data by running a series of experiments where temperature is varied. For example, a series of experiments was run where the same five concentrations of 4-carboxybenzene sulfonamide where injected at five different temperatures (15°, 20°, 25°, 30° and 35° C).

In the figure shown here, it is interesting to note that the shape of responses changes significantly as the temperature is increased from 20° to 35° (only one concentration response is shown).

Single concentration responses at two temperatures. The association and dissociation rates both are affected by temperature.

Global fits of the responses at all the temperatures were done and KD values obtained (responses not shown). A Van’t Hoff plot was then done to obtain the enthalpy value ΔH for the binding interaction.



Van’t Hoff Plot for biomolecular interaction of carbonic anhydrase with 4-CBS. Responses are from 5 different temperatures.

We hope you enjoy this column and return regularly for future posts, which will provide additional tips for when you use Reichert systems to perform SPR. You can read more about SPR elsewhere on Reichert’s website, including our first blog post and an earlier blog on performing kinetic and equilibrium analyses.

We also ask you to provide your own input and suggestions to make this column even better. Contact us if you have any questions or topics you would like to discuss, or if you have certain tips of your own that you would like to share.