Mary Murphy Ph.D. Applications Scientist Reichert Technologies, a unit of Ametek
Resolving Nonspecific Binding, Regeneration and Other Problems
Surface plasmon resonance, a label-free, real-time way to examine protein binding and other molecular interactions, is getting easier as manufacturers have streamlined instruments and supporting software. But problems can still arise. Here are some common issues and suggestions to solve them:
Inactive targets—Your target protein may have become inactive, or may be showing low binding activity to your analyte. This may not be due to your protein, but because the binding pocket on the target could be close to a primary amine bound to the sensor chip surface, making it less accessible. Try coupling the target to the slide differently. For example, perform a capture experiment instead of a covalent coupling experiment, or couple the protein and analyte via a thiol group.
Nonspecific binding—This happens when analytes bind to the SPR surface instead of just to the target. Your results will make this binding appear stronger than it actually is. Minimize this effect by supplementing your running buffer with additives like a surfactant or bovine serum albumin (BSA), or add dextran or polyethylene glycol (PEG) if appropriate. Another approach is coupling a compound that doesn’t bind the analyte on the reference. Still another approach is to change your sensor chip type.
Negative binding signals—Sometimes you’ll see a binding signal, but it appears that your analyte binds to your reference more strongly than to your target. This can be caused by a buffer mismatch, volume exclusion, or nonspecific interactions. There are a number of ways to resolve this, starting with the approaches covered in “nonspecific binding” above. Other approaches include testing the suitability of your reference channel, by injecting the highest concentration of your analyte over a native surface, over a deactivated surface, and over a BSA or IgG surface.
Regeneration problems—To use your sensor chip surface again, you must remove the analyte but keep the ligand intact. Because the physical forces behind your experimental binding are often unknown, successful regeneration is usually an experiment in itself! You’ll need to identify the appropriate solution to regenerate the sensor surface, so the chip can be reused for multiple analyte injections. There are a number of different solutions available, including acidic solutions (10 mM glycine pH 2, or 10 mM phosphoric acid), basic solutions (10 mM NaOH), or high salt solutions (2 M NaCl) to test. Also, adding 10% glycerol can be helpful for target stability.
SPR is a powerful technique for measuring biomolecular interactions, combining microfluidics, surface chemistry, and the surface plasmon resonance phenomenon to monitor label-free interactions. Knowing what common problems look like as well as ways to resolve them will help produce more reliable, valuable data. You can learn more about these troubleshooting techniques here and here.