Technical Notes

2 TECHNICAL NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. optimization experiments. Although standard procedures for immobilization are well-established for certain types of proteins, the general process is time-consuming and bears the risk of irreversibly damaging the ligand on the chip surface, thereby wasting costly consumables. Figure 1 Optimizing ligand immobilization to a SPR biosensor chip by testing different buffer conditions. Lower pH buffer conditions provide better conditions for immobilization of ligand to the SPR biosensor chip as determined by pH scouting experiments. The larger the resonance units (RU) value measured upon injection of a ligand solution, the better accumulation of the ligand on the chip surface due to electrostatic interactions. Buffers ranging in pH from 4.0 to 5.5 were tested.In this example, pH 4.0 – pH 5.0 are in principle suited for coupling of the ligand since they trigger a sufficient accumulation. In contrast, pH 5.5 is not suited for immobilization. Here, we demonstrate how a quick thermal stability screening of immobilization conditions for SPR assays can dramatically accelerate the assay development procedure. Therefore, optimizing immobilization conditions o en represents an early bottleneck in SPR assay development since it can damage the ligand during coupling, resulting in a non-functional biosensor surface and even a wasted biosensor chip. Procedures such as "pH-scouting", or "pre- concentration" are used to identify immobilization conditions which trigger sufficient surface attachment of the ligand (Figure 1). In these experiments, different immobilization buffers with varying, low pH-values containing the ligand are injected into the flow cell of the biosensor, and the accumulation of ligand on the sensor surface is monitored over time. This procedure typically takes 1-2 hours to prepare and perform, and identifies immobilization conditions without providing information on the functionality of the immobilized protein. If pH treatment conditions used for immobilization result in unfolding of the protein (partial or complete), this could render it inactive. This is only revealed a er performing subsequent binding experiments and getting negative or questionable results. Ultimately, this results in discarding the biosensor chip and repeating

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