Application Notes

9 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. by centrifugation 72 h post infection and stored at -80 °C until use. Cell membranes were prepared by thawing the pellet of 1 L expression culture in 40 mL hypotonic buffer containing 10 mM MES pH 6.0, 10 mM MgCl 2 , 20 mM KCl and EDTA-free complete protease inhibitor cocktail (Roche). Cells were broken with a Polytron PT1300D (Kinematica), filled up to 300 mL with high salt buffer containing 50 mM MES pH 6.0, 1000 mM NaCl and centrifuged at 45.000 RPM in a Ti45 rotor (Beckman Coulter) for 45 min. Purified membranes were resuspended in buffer containing 10 mM MES pH 6.0, 10 mM MgCl 2 , 20 mM KCl and 40 % Glycerol (w/v) and stored at -80 °C until further use. Membranes were thawed on ice and solubilized in 50 mM MES pH 6.0, 800 mM NaCl, 10 % Glycerol (w/v), 1 % (w/v) Lauryl Maltose Neopentyl Glycol-3 (LMNG-3) shaking smoothly 3 h at 4 °C. Unsolubilized material was removed by centrifugation at 150.000xg for 45 min at 4 °C. The supernatant was incubated with TALON IMAC resin (Clontech) over night. The resin was washed with 20 CV of 50 mM MES pH 6.0, 800 mM NaCl, 10 % Glycerol, 50 mM Imidazole, 0.02 % LMNG. Bound receptor was eluted with 50 mM MES pH 6.0, 800 mM NaCl, 10 % Glycerol (w/v), 300 mM Imidazole, 0.02 % LMNG-3 in a minimal volume. Purified receptor was buffer exchanged on a PD10 column (GE Healthcare) into 50 mM MES pH 6.0, 800 mM NaCl, 10 % Glycerol (w/v), 0.02 % LMNG- 3, and concentrated with 100 kDa MWCO Vivaspin concentrators (Sartorius) to 1.3 mg /mL. Thermal stability screen A2A-BRIL ligand stability screen Samples were prepared in 96-well PCR plates on ice. The assay was carried out at a protein concentration of 0.20 mg /mL. Therefore, 2.5 µL of purified A2A-BRIL at 3.0 mg /mL were diluted with buffer containing 50 mM HEPES pH 7.5, 800 mM NaCl, 10 % Glycerol (w/v), 0.01 %LMNG-3/0.002 % CHS into a final volume of 33.75 µL. 3.75 µL of ligand stock solution (1 mM in DMSO) was added and samples were mixed thoroughly. Samples were manually loaded into nanoDSF Grade Standard Capillaries (NanoTemper Technologies) in triplicates and transferred to a Prometheus NT.48 nanoDSF device (NanoTemper Technologies). Thermal unfolding was detected during heating in a linear thermal ramp (2.5 °C/min; 20 °C to 90 °C) with an excitation power of 80 %. Unfolding transition points were determined from changes in the emission wavelengths of tryptophan fluorescence at 330 nm, 350 nm and their ratios. Data was analyzed with the Prometheus PR. Control so ware (NanoTemper Technologies). Test-GPCR buffer stability screen Samples were prepared in 96-well PCR plates on ice. The assay was carried out at a protein concentration of 0.26 mg /mL. Therefore, 6.0 µL of purified test-GPCR at 1.5 mg /mL were diluted with buffer containing 5 mM MES pH 6.0, 800 mM NaCl, 10 % Glycerol, 0.02 %LMNG-3 into a final volume of 26.25 µL. 8.75 µL of the Solubility & Stability Screen 2 (Hampton Research) was added and samples were mixed thoroughly. Only wells A1-A12, F1-F12, G1-G12 and H1-H12 of the Solubility & Stability Screen 2 were used. Samples

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