Technical Notes

6 TECHNICAL NOTE ©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. The resulting large Reference Area makes the SQ value insignificant. Upon further examination, it was revealed that most buffer conditions containing TCEP resulted in G9a aggregation (data not shown). Discussion Using the Buffer Exploration Kit, 96 buffer conditions were tested for the highest SQ value which, by definition points to the condition with the highest assay sensitivity and reproducibility. The model used was the molecular interaction between G9a and SAM using Dianthus NT.23PicoDuo. We found that the SQ is dependent on buffer condition, and that we could select conditions with the highest signal amplitude and reproducibility. The two-part layout of the buffer screen (i.e. le side of the plate filled with variations of buffers, salt, and detergents vs. the right side filled with a variety of additives), allowed the simultaneous analysis of buffer conditions with and without additives. For the interaction between G9a and SAM, a positive impact on the SQ was shown with the addition of MgCl 2 and CaCl 2 , while the presence of EDTA caused the SQ to deteriorate. In addition, the stability of the reference and positive control could be tested in different buffers, with aggregation of the unbound G9a could be observed in conditions containing 1 mM TCEP. Rapid testing of 96 different conditions with the Buffer Exploration Kit enables assay development for target proteins that are less well studied and understood. This systematic approach provides valuable insight on the stability of the target molecule but also a deeper understanding of ligand interactions. The results presented suggest, for example, that G9a aggregates in the presence of TCEP and also that divalent cations mediate or promote its interaction with SAM. The strategy described here provides an efficient and data-rich approach to assay development. Setup and data analysis was designed with ultimate usability in mind. First, the process has a streamlined setup thanks to the Buffer Screen assay template provided in the DI.Control So ware. Second, the intuitive heatmap visualization of clustered conditions allows for quick and easy interpretation. Third, quantitative evaluation of results is made convenient as SQ values are automatically calculated by DI.Control so ware. Lastly, this strategy can be fully automated for the shortest possible runtime (5 hours for this study) with employing commercially available liquid handling systems. Materials and methods G9a was labelled with the RED-NHS 2nd Generation labelling kit (Cat# NT-111, NanoTemper Technologies) following the kit protocol . A er labelling, the protein was eluted and stored in 50 mM HEPES pH 8.0, 150 mM NaCl, 2 mM Glutathione (GSH), 0.005 % Tween®20, which is referred to as the initial sample buffer. In order to test the different assay conditions, a binding check (comparison of reference and positive control) was performed for each of the 96 buffers contained in the Buffer Exploration Kit (Cat# NT-B001, NanoTemper Technologies). First, two stock solutions, one for the reference and

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