6
APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
We were able to obtain melting curves for the
test-GPCR in nanoDSF experiments (Figure 6) and
used the Prometheus NT.48 system (NanoTemper
Technologies) to further characterize the thermal
stability of the test-GPCR in presence of 22 selected
compounds (Table 2) and diff erent buff er systems
(Table 3).
The selected compounds have µM to nM aff inity
towards human test-GPCR in the functional Ca
2+
-
FLIPR assays. IC50 values obtained from Ca
2+
-FLIPR
assays are reported in Table 2. The rank order of
compounds with sub-µM aff inity in the calcium
reporter assay and strong ∆T
m
shi in nanoDSF assay
are in good correlation (Table 2). As expected, high
µM aff inity compounds do not have a pronounced
stability eff ect on the purifi ed receptors (Figure 7
and Table 2), whereas three out of fi ve high aff inity
Figure 6: Typical nanoDSF unfolding curves using test-GPCR. Recordings of tryptophan fl uorescence at 330 nm are shown in the top half of the graph
and the corresponding fi rst derivative is plotted in the bottom half. Infl ection points (equivalent to the T
m
) are shown as vertical lines.
Figure 7: Summary of thermal stabilization of test-GPCR by selected compounds
(see also Table 2).
