Surface Plasmon Resonance (SPR) provides important information about how biomolecules interact. Information on association and dissociation kinetics and affinity can be obtained directly in real-time and label-free. This type of experimentation is very useful for researchers because generally, all classes of biomolecules can be studied and experiments can be run in an automated manner once optimum conditions are determined. But the determination of optimum conditions can sometimes be problematic. Here are some common challenges researchers encounter when using the technique and how to overcome them.
Three of the biggest challenges encountered are (1) lack of target activity once it is coupled, (2) non-specific binding and (3) regeneration problems.
1. Target Can Become Inactive
The target may become inactive or only exhibit minimal activity when coupled to the sensor chip surface, particularly when doing amine coupling. The building blocks of proteins are amino acids and amino acids have primary amine functional groups. Therefore, there are always multiple points on each protein/target that can be coupled to a sensor chip surface via amine coupling. If the binding pocket on the target is close to a primary amine that is bound to the sensor chip surface, it is then less accessible to bind to analyte. This can result in responses that may be much smaller than expected (or in the worst case, no binding is seen). The other issue with direct coupling is that a lower pH is needed to couple the protein (1 to 2 units below the pI). Dissolving the protein in a lower pH buffer could potentially denature the protein making it lose its primary structure and hinder binding.
- Couple the target to the slide differently. If the target has available, free thiol groups (-SH) try thiol coupling. Since proteins have fewer thiol groups (some have none), thiol coupling is more selective. Maleimide or aldehyde groups on the target can also be coupled which also adds to the selectivity (note: these other types of experiments are much less common than amine coupling (most common) and thiol coupling).
- Do a capture experiment instead of a covalent coupling experiment. Capture the protein onto the sensor chip via a tag. Since the protein is captured over the surface in running buffer, the possibility of denaturation at lower pH values is eliminated. Also advantageous is that, in most cases, denaturation from regeneration is not an issue because the entire protein-analyte interaction pair is removed with each regeneration. The protein is then captured anew prior to the next analyte injection. Another advantage is the tag can usually be minimally added at different points on the target, allowing a choice of exactly where the target will be captured.
- Couple the target-analyte binding pair to the surface. As long as the interaction is reversible, it can help keep the binding pocket available and stabilized for further experimentation. An excess of the analyte is needed to do this.
2. Non-Specific Binding
Non-specific binding can be a major problem. In a perfect world, the non-binding analog of the protein being coupled would be available for binding on the reference surface. This would make the sample and reference surfaces the same and only the specific binding would be seen when the sample responses were reference subtracted. This is rarely the case with real experiments since most researchers do not have non-binding analogs available. For covalent coupling experiments, the reference surface is normally treated the same as the sample surface but without the protein being coupled. This means the slide surface is more available for non-specific binding on the reference than the sample channels.
- Minimize non-specific binding by supplementing running buffer with certain additives. Some common additives that can be used include: surfactant, such as Tween-20 at a concentration of 0.005 percent to 0.1 percent, sodium chloride up to 500 mM and Bovine serum albumin at concentrations of 0.5 to 2 mg/ml. Or, if using a carboxymethyl dextran chip, 1 mg/ml of carboxymethyl dextran can be added to the running buffer. Or, for a planar COOH sensor chip with polyethylene glycol add 1 mg/ml polyethylene glycol to the running buffer.
- If analyzing a positively charged analyte, block the sensor chip with ethylenediamine instead of ethanolamine after amine coupling. This will reduce the negative charge on the sensor surface and thus decrease the potential for non-specific binding.
- Couple a compound that does not bind to your analyte on the reference channel.
- If the slide being used shows too much non-specific binding, try a different type of slide (e.g. Try a planar slide if you see a lot of non-specific binding using dextran.)
3. Regeneration Problems
Regeneration can at times be very difficult. This is a challenge because poor choice can cause denaturation and loss of protein activity. Sometimes the interaction is just very difficult to even break up. Or, the regeneration may go well for a couple of injections, then start to fail. There are three major types of regeneration solutions: Acid, Base and Ionic. The choice of which to use depends on the type of interaction between the binding pair and on the stability of the target when exposed to each type of solution. One should always use the weakest regeneration solution (lowest concentration) that completely removes the analyte (meaning the baseline returns to where it started before the analyte was injected). Even with the most careful choice of regeneration solution, there can still be issues with denaturation of the target and loss of activity. Without good replicates (being able to get the same response when injecting the same concentration of analyte multiple times) good kinetics cannot be obtained since the expected maximum response needed for kinetics equations keeps changing.
- Add glycerol until it comprises 5 percent to 10 percent of a regeneration solution. This increases the probability of the target on the slide surface staying active. For example, a 9:1 solution of 10 mM glycine pH 2.0: glycerol can preserve full activity of an antibody target while regenerating the chip sensor surface completely. Without glycerol, the regeneration solution denatures some of the immobilized antibody.
- Inject low to high concentrations of analyte over the target, (association/dissociation) without regeneration.
- Inject a single high concentration and create a gradient so that a mixture of concentrations is generated from one injection
While SPR experimentation can be challenging, handling the three biggest challenges discussed here is essential to developing optimum conditions for robust SPR analysis methods.
Filed Under: Drug Discovery