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APPLICATION NOTE 10 ©2019 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 2% DMSO. Each fragment dilution was then mixed with equal volumes of labeled G9a containing assay buffer, resulting in final concentrations of 1% DMSO and 5 nM labeled G9a. In all experiments with SAM as a ligand, a final amount of 1% DMSO was added to mimic the conditions for fragment dilutions. A er the 384-well microplates were loaded with the fragment + G9a mix, they were equilibrated for 2.5 hr and centrifuged for 30 sec at 1000 x g before loading onto Dianthus NT.23PicoDuo. The system was set to 20°C as set temperature. The samples were first measured for 1 sec without heating and for 5 sec with the IR-laser turned on. The two optical systems in Dianthus were used in parallel, resulting in an overall measurement time of ~30 min per plate. Measured fluorescence values collected are displayed as relative fluorescence, where the fluorescence obtained at ambient temperature is normalized to one, and as normalized fluorescence (Fnorm) which describes the ratio between fluorescence values (F1) a er and the fluorescence values (F0) prior to IR laser activation and is typically given in ‰. The dissociation constant or K d , is obtained by fitting a dose-response curve to a plot of Fnorm vs. ligand concentration. References 1. Scannell JW., et al. (2012) Diagnosing the decline in pharmaceutical R&D efficiency. Nature Reviews Drug Discovery. 11(3),191-200. 2. Gupta, Amit J.; Duhr, Stefan; Baaske, P. (2018). Microscale Thermophoresis (MST). Encycl. Biophys. Living Edi. 3. Soumyanarayanan, U., and Dymock, B.W. (2016). Recently discovered EZH2 and EHMT2 (G9a) inhibitors. Future Med. Chem. 8, 1635–1654. 4. Liu, Q., and Wang, M.-W. (2016). Histone lysine methyltransferases as anti-cancer targets for drug discovery. Acta Pharmacol. Sin. 37, 1273–1280. 5. Ackloo, S., Brown, P.J., and Müller, S. (2017). Chemical probes targeting epigenetic proteins: Applications beyond oncology. Epigenetics. 12, 378–400. 6. Bertz, S.H. (1981). The first general index of molecular complexity. J. Am. Chem. Soc. 103, 3599–3601. 7. Hendrickson, J.B., Huang, P., and Toczko, A.G. (1987). Molecular complexity: a simplified formula adapted to individual atoms. J. Chem. Inf. Model. 27, 63–67. 8. Jayaram, H., Hoelper, D., Jain, S.U., Cantone, N., Lundgren, S.M., Poy, F., Allis, C.D., Cummings, R., Bellon, S., and Lewis, P.W. (2016). S-adenosyl methionine is necessary for inhibition of the methyltransferase G9a by the lysine 9 to methionine mutation on histone H3. Proc. Natl. Acad. Sci. USA. 113, 6182–6187. NT-DI-001-02