Figure 8. Differential scanning fluorimetry of Ric-8A and its mutants.
Samples (10 μl) of wild type Ric-8A(1–491) and mutants thereof (~1 mg/ml) in 50 mM HEPES, pH 8.0, 150 mM NaCl and 1 mM TCEP were dispensed into glass capillaries and placed into the sample chamber of a Prometheus NT.48 differential scanning fluorimeter (NanoTemper Technologies, Inc, Munich, Germany). Samples were subjected to a time-dependent temperature gradient over 20–75°C at a rate of 1°C/min. Fluorescence emission at 330 nm and 350 mn (excitation wavelength, 295 nm) was recorded at seven second intervals. The transition temperature for thermal denaturation (Tm) is defined as the temperature at the maximum the first derivative of the ratio of fluorescence emission at 350 and 330 nm (F350/F330) as determined by a polynomial fit to the temperature-fluorescence ratio curve implemented in the manufacturer’s software.