Application Notes

8 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Material and Methods Protein preparation A2A-BRIL A2A-BRIL was prepared as previously described [5]. In brief, Sf9 cells in SF-4 Baculo Express ICM (BioConcept) at a density of 2.0x10 6 /mL were infected with high-titer recombinant Baculovirus (FlashBac system) at MOI (multiplicity of infection) of 10. Cells were harvested by centrifugation 72 h post infection and stored at -80 °C until use. Cell membranes were prepared by thawing the pellet in hypotonic buffer containing 10 mM HEPES pH 7.5, 10 mM MgCl 2 , 20 mM KCl and EDTA-free complete protease inhibitor cocktail (Roche). Cells were broken with a Polytron PT1300D (Kinematica) and centrifuged at 45.000 RPM in a Ti45 rotor (Beckman Coulter) for 45 min. Membranes were washed extensively by repeating this step 2-3 times with hypotonic buffer, followed by 2-3 washing steps using a high osmotic buffer containing 10 mM HEPES pH 7.5, 1000 mM NaCl, 10 mM MgCl 2 , 20 mM KCl and EDTA-free complete protease inhibitor cocktail (Roche). Purified membranes were resuspended in buffer containing 10 mM HEPES pH 7.5, 10 mM MgCl 2 , 20 mM KCl and 40 % Glycerol (w/v) and stored at -80 °C until further use. Membranes were thawed in the presence of 4 mM Theophylline and 2.0 mg /mL iodoacetamide. A er 30 min incubation on ice, membranes were solubilized in 50 mM HEPES pH 7.5, 800 mM NaCl, 10 % Glycerol (w/v), 1 % (w/v) Lauryl Maltose Neopentyl Glycol-3 (LMNG-3) / 0.2 % (w/v) cholesteryl hemisuccinate (CHS) and EDTA-free complete protease inhibitor cocktail (Roche) by stirring for 2 h at 4 °C. Unsolubilized material was removed by centrifugation at 150.000 xg for 45 min at 4 °C. Imidazole was added to the supernatant to a final concentration of 20 mM and incubated with TALON IMAC resin (Clontech) over night. The resin was washed with 10 column volumes (CV) of 50 mM HEPES pH 7.5, 800 mM NaCl, 10 % Glycerol (w/v), 20 mM Imidazole, 0.01 % LMNG-3/0.002 % CHS (w/v) and 2 mM Theophylline, 5 CV of 50 mM HEPES pH 7.5, 800 mM NaCl, 10 % Glycerol (w/v), 25 mM Imidazole, 0.01 % LMNG-3/0.002 % CHS (w/v) and 2 mM Theophylline, and 2 CV of 50 mM HEPES pH 7.5, 800 mM NaCl, 10 % Glycerol, 50 mM Imidazole, 0.01 % LMNG-3/0.002 % CHS (w/v) and 2 mM Theophylline. Bound receptor was eluted with 50 mM HEPES pH 7.5, 800 mM NaCl, 10 % Glycerol (w/v), 300 mM Imidazole, 0.01 % LMNG-3/0.002 % (w/v) CHS and 2 mM Theophylline in a minimal volume. Purified receptor was buffer exchanged on a PD10 column (GE Healthcare) into 50 mM MES pH 7.5, 800 mM NaCl, 10 % Glycerol (w/v), 0.01 % LMNG-3/0.002 % CHS (w/v), 2 mM Theophylline and concentrated with 100 kDa MWCO Vivaspin concentrators (Sartorius) to 3 mg /mL. Test-GPCR Test-GPCR was prepared similar to A2A-BRIL. In brief, Sf9 cells in SF-4 Baculo Express ICM (BioConcept) at a density of 2.0x10 6 /mL were infected with high-titer recombinant Baculovirus (FlashBac system) at MOI (multiplicity of infection) of 10. Cells were harvested

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