Technical Notes

2 TECHNICAL NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Results In these experiments, we demonstrate the utility of Tycho NT.6 for monitoring protein quality during a purification workflow. Thermal unfolding profiling and thermal shi functionality tests were used throughout the purification of an INO80 chromatin remodeler sub-complex (HSAX), consisting of the INO80 HSA domain (INO80 HSA , from hereon referred to as HSA), actin, and two actin-related proteins, Arp4 and Arp8 (Figure 1A) 1,2 . A er initial purification using a C-terminal Strep-tag, the complex was further purified using size-exclusion chromatography (SEC) with a S200 SEC column. The elution profile showed two clearly distinct elution peaks (Figure 1B). A er running SDS-PAGE analysis, peak F3 was shown to contain all 4 proteins of the complex, indicated by 4 different bands with the correct molecular weight. In contrast, fraction G7 mainly contained one protein, likely Arp8 which has the highest molecular weight (Figure 1C). Analysis of the main peak fractions with the Tycho NT.6 and comparison of the obtained unfolding profiles with a reference sample of previously purified HSAX unambiguously identified fraction F3 as the fraction containing properly assembled HSAX (Figure 1D). These experiments were performed much faster and efficiently when compared to traditional SDS- PAGE. Fraction G7 showed a distinct unfolding profile different from either fraction F3 or the reference, corroborating that this sample does not contain the intact HSAX complex. every time for new experiments. While standard operating procedures and protocols can be created to provide consistency and confidence in the sample preparations, it is important to rigorously test the quality and functionality of samples before committing valuable time and expense to performing more detailed characterizations. Quality tests are o en overlooked as they can be time-consuming and require separate workflows which have to be performed in parallel. Functionality of a protein (or lack thereof) is typically evaluated long a er the protein purification procedure is completed, or not at all, potentially wasting researchers' precious time and translating into inconclusive or non-reproducible results. Tycho NT.6 is a simple label-free platform that provides quick protein quality validation. It can be introduced with ease at virtually every step of the protein purification workflow a er cell lysis and initial purification to final production. Tycho NT.6 generates thermal unfolding profiles of protein samples, which are instrumental to evaluate the quality and conformational integrity of proteins. In addition, Tycho NT.6 can monitor and detect binding events by ligand-induced changes in the unfolding profiles. Thus, the quality, stability and functionality of a protein can be addressed within a quick, three- minute experiment.

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