Reducing Non-Specific Binding
When running a Surface Plasmon Resonance (SPR) experiment to characterize the interaction between two molecules, you may need to take steps to minimize non-specific binding. This Surface Plasmon Resonance Insider post will provide you with the information you need to minimize non-specific binding when it interferes with a binding interaction under study.
Non-specific binding occurs when the analyte interacts with the surface of the sensor chip and can be verified from the response on the reference channel. The measured response on the sample channel is the sum of the specific binding, any non-specific binding and the bulk refractive index shift if present (see Figure 1) while the response on the reference channel is soley from non-specific binding and any bulk refractive index shift. If the response on the reference channel is greater than a third of the sample channel response, the non-specific binding contribution should be reduced.
Figure 1: Impact of Non-specific binding on experimental results.
You can 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% to 0.1%.
- · NaCl up to 500 mM.
- · Bovine serum albumin (BSA) at concentrations of 0.5 to 2 mg/ml.
- · If you are using a carboxymethyl dextran chip, you can add 1 mg/ml of carboxymethyl dextran to the running buffer.
- · If you are using a planar COOH sensor chip with polyethylene glycol (PEG), then you can add 1 mg/ml PEG to the running buffer.
- · If you are analyzing a positively charged analyte, you can block the sensor chip with ethylenediamine instead of ethanol amine after amine coupling. This will reduce the negative charge of the sensor surface and thus decrease the potential for non-specific binding.
You can also reduce non-specific binding by coupling a compound that does not bind to your analyte on the reference channel. Or, if the slide you are using 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, etc.
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 our previous blog entitled “Choosing the Right Chip”.
If you would like advice on determining the most running buffer 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.