The next two posts will provide tips related to data analysis – what types of analysis you can do and what type of data is needed to get the results you want.
1) When doing Surface Plasmon Resonance data analysis, global fits are preferred. If you are unable to fit the data globally, there may be a stability problem with the target or analyte, or there may have been a dilution error somewhere in the sample preparation.
In your experimental plan, be sure to do replicate injections at each concentration. Replicate injections provide useful information about the stability of the surface with time. In addition, if regeneration is needed, replicates show that the magnitude and shape of the responses is not affected.by the regeneration solution. The following example shows the kinetic fit for 3-carboxybenzenesulfonamide binding to carbonic anhydrase. Replicate injections overlay for each of six concentrations and a good global fit is obtained.
Kinetic fit for 6 concentrations of 4-carboxybenzenesulfonamide (4-CBS) binding to carbonic anhydrase - fit to a 1:1 binding model in Tracedrawer. The KD value obtained is 892 nM.
2) When doing a global fit of Surface Plasmon Resonance sensorgrams, pay close attention to the residuals. Residuals are the difference between the sample responses and the fit. The best global fit has minimal and random residuals. In the following example, we used a 1:1 binding model in Tracedrawer to globally fit six concentrations of antigen binding to antibody (sample responses in various colors, black lines show fit). The residuals are random and minimal which speaks to the good quality of the fit.
Responses for 6 HSA concentrations injected in duplicate and fit to 1:1 binding model in Tracedrawer. HSA binding is to Anti-HSA.
The KD value obtained is 5.34 nM.
Residuals – difference between data and fit form the above responses.
3) If the majority of the sensorgrams are at equilibrium, and you need to determine just the equilibrium dissociation constant (KD), you can do a Langmuir Isotherm plot. To do this, choose a point or interval near the end of the association part of your injections and plot the responses versus concentration. For the example of 4-carboxybenzenesulfonamide binding to carbonic anhydrase, the following Langmuir Isotherm plot was obtained in Tracedrawer.
The maximum responses for 6 concentrations of 4-CBS binding to carbonic anhydrase are plotted vs concentration to obtain a KD value. The KD value obtained is 809 nM.
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.