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is checked a er incubation of the sample at diff erent
temperatures in native-PAGE. A er 30 min incubation,
human test-GPCR demonstrated half aggregation
at approximately 40 °C (apo) and 60 °C (#22 tool
compound for human) (Figure 5), respectively.
of the human isoform which are required for CPM
fl uorescence probe reactivity could be one reason for
the lack of melting curves in the CPM-DSF assay. We
used a native-PAGE unfolding assay as a reference
point for the thermal stability of human test-GPCR. In
this assay, protein oligomerization and aggregation
Figure 5: Comparison of human and rat test-GPCR. Upper panel: Complementary native PAGE stability assay for ligand screening using human and rat
test-GPCR; CPM-DSF is not working for the human isoform. Lower panel: Analysis of monomeric band thermal aggregation in native PAGE. Apoprotein
band insensitivity (marked as "") at 4 °C was used as a reference (100 %). Samples were incubated 30 min on ice, at 40 °C, 60 °C or 80 °C with and
without high aff inity ligand prior to analysis.
