Understanding how molecules interact is the objective of many research pharmaceutical projects. Not only it is key for the development of new drugs candidates, but also to elucidate the role these interactions play in all biological processes and cellular pathways.
Over the years, several biophysical tools have been developed to characterize interactions between proteins and other molecules. They can be divided into two categories based on how the binding partners are presented during the measurement: free in solution or immobilized.
In contrast to free in solution assays﹣where both binding partners are prepared in a solution of optimized buffer﹣immobilization requires one of the binding partners to be attached to a solid surface, while the other is flowed over the solid surface utilizing microfluidics. It is well-known that immobilization can have an impact on binding affinity and kinetics measurements.
Here are 4 effects immobilization can have on your binding affinity experiments.
1. Conformational changes of proteins
Proteins need to be in their native conformation in order to interact with their ligands. Immobilization, however, can cause conformational changes, thereby altering the protein’s interaction capabilities and compromising the success of a binding assay.
2. Binding interaction due to random orientation
In order for a binding interaction to take place, the binding partners need to be properly oriented. This means that when proteins are immobilized, they should be oriented on the surface in such a way that the binding sites are exposed to the analyte. However certain immobilization techniques are susceptible to the random orientation of the ligand to the surface. This random orientation can block binding sites and as a result, reduce the number of available binding sites which can lead to experimental irreproducibility. The two most common solutions to this problem are unidirectional immobilization and affinity capturing.
3. Non-specific binding of analytes to immobilization surface
Besides binding to the target protein, analytes may bind to the immobilization surface. This non-specific binding contributes to false positive results on a binding assay. Despite several strategies that can be implemented to reduce this unwanted effect, its complete elimination may not be possible given the constraints of each experiment.
4. Increased costs and timing of each experiment
Immobilization of proteins requires finding the right conditions for each experimental run, which includes avoiding the effects described above and involves, among other things, having to run multiple controls alongside the samples. This time consuming and costly optimization procedure is often a bottleneck in surface-based approaches.
So, if you’re deciding between different methods for measuring molecular interactions, make sure you take into account how your binding assay can be affected by your method of choice. By doing this, you’ll be able to make a thoughtful and more informed decision.