Thermal Denaturation (Thermofluor) Assay

The Thermofluor (or Differential Scanning Fluorescence) Assay, used to analyze protein thermal stability, was performed as previously described [89–91]. Briefly, purified TBCD•ARL2•β-tubulin (5 μM) was pre-incubated with a 10-fold molar excess of GDP, GTPγS or no nucleotide for one hr on ice. MgCl₂ was included at a final concentration of 1 mM to facilitate nucleotide binding. Immediately prior to analysis, 2 μL of a 1:100 dilution of SYPRO Orange dye (Sigma, catalog no. S5692) was added to 20 μL of each sample. Fluorescence intensity (Ex 488 nm, Em 603 nm) was monitored, as a measure of thermal denaturation, using a StepOnePlus Real-Time PCR System (ThermoFisher, catalog no. 4376600) every 2 min over the range of 25–55 °C (0.5 °C/min temperature gradient). We fit the resulting curves to a Boltzmann sigmoidal equation using GraphPad Prism software to determine the melting temperature (inflection point) for each sample. Data represent the averages of two experiments for each sample.