10
APPLICATION NOTE
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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 75 %. Unfolding transition points
were determined from changes in the emission
wavelengths of tryptophan fl uorescence at 330 nm,
350 nm and their ratios. Data was analyzed with
the Prometheus PR. Control so ware (NanoTemper
Technologies).
Acknowledgments
We thank Dr. Nina Gommermann (NIBR, Novartis)
for producing various compounds used in this study
and Dr. Felix Freuler (NIBR, Novartis) for molecular
biology support. Dr. Andrew Quigley, Jit Ang and
Prof. Liz Carpenter for discussions and the possibility
to analyze samples using an instrument in the SGC
(Structural Genomics Consortium, University of
Oxford). We thank Dr. Sandra Jacob and Dr. Christine
Genick for useful discussions.
Tables
Table 1. Reference and commercial compounds screened using A2A-BRIL.
1
Strongly autofl uorescent compound
2
Reference without compound
Table 2. Blinded compound screen for test-GPCR.
1
Reference without compound
2
Classifi cation: 1: IC50 less than 100 nM. 2: IC50 between 101 nM and 1000 nM. 3:
IC50 over 1 µM
