Melting Temperature (Tm)
PCR , SYPRO Orange , Thermal Denaturation , Differential Scanning Fluorimetry (DSF)
Dulbecco’s phosphate-buffered saline
30-94 °C at 0.06 °C/s
excitation at 470 nm and emission at 610 nm
Differential scanning fluorimetry (DSF) was used to determine the melting temperatures (Tm) of the parental and mutant A26.8 variants. DSF was carried out in a Rotor-Gene 6000 real-time PCR instrument (Corbett Life Science, Mortlake, NSW, Australia). Samples were diluted in HyClone™ Dulbecco’s phosphate-buffered saline (D-PBS; GE Healthcare) at a final concentration, after mixing, of 0.33 mg/mL. A total volume of 30 μL in 0.2 mL thin wall PCR tubes (Axygen, Oneonta, NY) was used. SYPRO® Orange (Life Technologies, Burlington, ON, Canada) was diluted 1,000-fold from the 5,000x concentrated stock to the working dye solution in D-PBS and 15 μL were added to 15 μL of sample just prior to the experiment. Thermal denaturation was carried out by increasing the temperature from 30 °C to 94 °C at a rate of 0.06 °C/s. Fluorescence intensity, with excitation at 470 nm and emission at 610 nm, was collected at 1 °C intervals and analyzed with the Rotor Gene 6000 series software v1.7 (Corbett Life Science). The Tm values were determined from the peak of the first derivative transformation of the raw data. Each sample was measured in quadruplicate.