Figure 1. Non-specific binding vs specific binding of a protein analyte on a COOH coated SPR sensor chip with an immobilized ligand.
Non-specific binding is an important experimental parameter to control when using SPR systems. Non-specific binding is the binding of analyte to non-target molecules on the sensor surface, as illustrated in Figure 1 (all figures and tables can be found on the last page). The effect of non-specific interactions is a false positive contribution to the signal in a sensorgram. It is important for users to recognize non-specific binding and to implement strategies to reduce or eliminate its effects to get accurate kinetic data.
Non-specific binding is caused by molecular forces (charge-charge interactions, hydrophobic interactions, etc.) between the analyte and the sensor surface. To reduce and prevent nonspecific binding there are a number of experimental conditions that can be used. The most common methods include the addition of bovine serum albumin (BSA) as a blocking protein, the addition of a surfactant such as Tween 20, careful adjustment of the buffer pH, and the addition of salt. In this application note, a biological system demonstrating non-specific interactions is examined and various prevention methods are tested to evaluate their effectiveness. The system comprises rabbit IgG antibody as the model protein analyte, which interacts non specifically with a carboxylated gold sensor surface. The effects of different methods for reducing NSB of rabbit IgG are analyzed and explained. SPR users can use this applicate note to help determine which conditions to use to reduce NSB in their experiments.