Overcoming Regeneration Problems
Surface Plasmon Resonance (SPR) is the best technique for characterizing molecular interactions. One obstacle in carrying out a successful SPR experiment is identifying an appropriate solution to regenerate the sensor surface so the chip can be reused for multiple analyte injections. Unfortunately, it can be difficult to find the best solution to regenerate the sensor chip surface, and this “scouting” step can be very time-consuming. This Surface Plasmon Resonance Insider post will help you overcome some of these problems as you plan your SPR study.
Regenerating the sensor chip surface dissociates all of the analyte from the ligand, which allows you to use the same chip multiple times. Regeneration solutions need to be strong enough to completely dissociate the analyte from the ligand but not so strong that they will compromise or denature the ligand.
Typical regeneration solutions include acidic solutions (e.g., 10 mM glycine pH 2, or 10 mM phosphoric acid), basic solutions (e.g., 10 mM NaOH), high salt solutions (e.g., 2 M NaCl), or a combination of these conditions. We at Reichert have discovered that simply adding glycerol until it comprises 10% of a regeneration solution will greatly improve a regeneration solution’s utility. For example, a 9:1 solution of 10 mM glycine pH 2:glycerol was able to preserve full activity of an antibody ligand while regenerating the chip sensor surface completely. Without glycerol, the solution was found to denature some of the immobilized antibody.
The simple addition of glycerol will help you get the most out of your regeneration solution. Injecting multiple concentrations of analyte in duplicate or triplicate over the same ligand immobilized on a SPR chip results in very accurate measurements of equilibrium constants, kinetics rates and protein concentrations. Selecting a proper regeneration solution in the beginning of your SPR experiment saves time and cost by simplifying the “scouting” step, which ultimately conserves ligand and sensor chip usage.
We hope you found this post helpful and informative. You can read more about SPR elsewhere on Reichert’s website, including our first blog post and information on the technology of SPR.
If you would like advice on how to determine the best regeneration solution for your application, please do not hesitate to contact us. Also, please contact us if you would like to suggest topics for future blog posts or if you have any suggestions to make this column even better.