Technical Notes

3 TECHNICAL NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. By using Tycho NT.6, different buffers can be tested within minutes to unambiguously identify conditions under which the ligand protein is stable or when it unfolds, saving hours of experimentation testing a potentially unfolded or inactive protein sample and preventing the unnecessary costs and waste of biosensor chips. Results To demonstrate the versatility and applicability of Tycho NT.6 to improve SPR assay development, two different classes of proteins were selected. One protein was a kinase, which are popular drug targets and used in small molecule screenings. The other protein was a monoclonal IgG antibody (mAb), which are of interest as therapeutic molecules. The two proteins were resuspended in phosphate buffered saline (PBS) and tested in four standard immobilization buffers containing 10 mM acetate with pH values between 4.0 and 5.5. The kinase showed marked effects of the immobilization buffer on protein conformation (Figure 2A). The PBS sample used as a reference of properly folded protein showed a low initial ratio and a clear unfolding event that is temperature dependent, indicated by the inflection of the unfolding profile curve. At low pH (4.0 and 4.5), the kinase was entirely unfolded already at the start of the experiment, suggested by the very high initial ratio as well as the absence of an unfolding inflection. At pH 5.0, the initial ratio was intermediate, showing that the kinase was partially unfolded, and an unfolding event was visible, but shi ed to much lower temperatures compared to the PBS sample, signifying a destabilization of the kinase. Only pH 5.5 showed favourable conditions: the initial ratio was similar to the PBS sample, suggesting that the kinase was properly folded. These results suggest that pH 5.5 acetate buffer is the only buffer to be considered for immobilization tests for this kinase. In contrast to the kinase, the mAb showed no major effect of the immobilization buffer on conformational stability as indicated by similar initial fluorescence ratios of all samples.

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