One of the most widely used tools to detect and quantify molecular interactions is SPR. As with any methodology, some level of assay optimization is required as rarely does an assay work directly “out-of-the-box” the first time.
The key to a successful SPR assay is being able to efficiently and effectively immobilize one of the binding partners to a biosensor surface. With many factors affecting this process, finding optimum conditions can be time-consuming and potentially result in wasted sample and expensive consumables (biosensor chips). Is there an easier and faster way to optimize an SPR assay?
The challenge: pH getting in the way of a binding assay
In an SPR assay, typically one binding partner — the ligand — is covalently immobilized on the surface of a biosensor chip, while its binding partner — the analyte — is passed in an aqueous solution under a continuous flow rate to measure binding kinetics. Part of optimizing the assay includes determining the condition that will immobilize the most ligand to the chip.
This includes testing several buffer solutions of different pH values or ‘pH scouting’, a procedure that can take many hours to perform. Typically, low pH conditions are used to covalently immobilize the ligand. While this is good for covalent linkage, low pH can impede a protein’s ability to fold and/or bind appropriately. What is the point of the whole assay if the binding abilities of the ligand are lost, right?
The solution: ID’ing the best conditions for the molecule to behave
Optimizing an assay to characterize any biomolecule can be tricky especially if it’s immobilized onto a surface. Wouldn’t it be nice if you could screen for conditions that retained the target molecule’s functional activity while also achieving a high level of immobilization of the molecule to the chip?
In this technical note, learn how to more effectively screen and select an SPR immobilization buffer that will likely maintain the structural integrity of your protein sample. Do this as quickly as in 3 minutes so you can spend more time focused on getting the consistent and detailed binding results from your biosensor assay.