Surface plasmon resonance (SPR), a label-free, real-time way to examine protein binding and other molecular interactions, is getting easier as manufacturers have streamlined SPR instruments and supporting software. But problems can still arise.
Troubleshooting Your SPR Assay
Here are some common issues and suggestions to solve them:
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 itself—instead, 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 target via a thiol group.
Non-specific binding happens when analytes bind to the SPR surface instead of just to the target. This can make 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). You can also add dextran or polyethylene glycol (PEG), if appropriate. Another approach is coupling a compound that doesn’t bind the analyte on the reference. You can sometimes also change your sensor chip type.
Negative Binding Signals
Sometimes you’ll see a binding signal in your SPR assay, 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 other non-specific interactions. There are a number of ways to resolve this, starting with the approaches covered in “non-specific 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 in Your SPR Instrument
To successfully regenerate, you must either remove the analyte but keep the ligand intact (coupling experiment) or remove both the target and analyte each time (capture experiment). Because the physical forces behind your experimental binding may not be known, successful regeneration can be 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 Technology Provides Reliable and Valuable Data
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 when using SPR technology as well as ways to resolve them will help produce more reliable, valuable data.
Surface Plasmon Resonance (SPR) is the gold standard for measuring biomolecular binding without the need for labeling (i.e., label free detection of kinetics). SPR is especially valuable because it doesn’t just provide information at the start and end of a binding event, but can be used to follow association and dissociation kinetics of biomolecules in real-time. […]
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