Technical Notes

Monitoring the impact of storage-dependent denaturation on protein quality using Tycho NT.6

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TECHNICAL NOTE Tycho is a tra demark of NanoTemper Technologies GmbH, Munich, Germany. NanoTemper is a registered trademark of NanoTemper Technologies GmbH, Munich, Germany. ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. TE-TY-005-01 nanotempertech.com The effect of storage-related stress on the outcome of biophysical experiments was then evaluated by measuring the interaction of the p38 samples with the small molecule inhibitor SB239063 by MicroScale Thermophoresis (MST) (Figure 1B). Results of the MST analysis confirmed that the presence of unfolded protein in the sample directly influences T i (°C) Initial Ratio △ Ratio MST Kd (nM) MST amplitude (F norm ( 0 / 00 )) MST signal-to-noise Fresh 53.1 0.725 0.193 59 28.5 25.7 Freeze-thaw cycles 53.2 0.739 0.176 64 21.6 7.4 50% denatured 53.5 0.812 0.088 68 12.8 9.5 Fully denatured n.a. 0.852 0.025 n.a. 0 n.a. Table 1: Summary of Tycho NT.6 and Monolith analysis of p38 kinase under different storage conditions assay outcome. The MST binding amplitude decreased with the presence of increasing amounts of unfolded protein and the overall assay quality was lower, as indicated by greatly reduced signal-to- noise values (Table 1). Fully denatured p38 did not show an interaction. Conclusions We show that the Tycho NT.6 system can be used to quickly evaluate different storage conditions, such as freezing strategies, to provide a better insight on the quality of the proteins being tested. The results demonstrate that poor sample quality and the presence of denatured protein can directly influence assay performance and the experimental outcome. The Tycho NT.6 system is well-suited to swi ly validate protein quality in as short as 3 minutes, prior to performing further biophysical analysis which can be time-consuming and costly to run.

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