Sypro Orange (Invitrogen), supplied as a 5,000X solution in dimethylsulfoxide, was diluted in the buffer solution (1:1000) and mixed well just prior to preparing samples for the assay. Reactions of 30 μL were prepared at room temperature and delivered to 96-well optical plates (Applied Biosystems) before sealing with optical film. To the protein solutions at a final concentration of 0.5–1.5 μM in Buffer B, 3 μL of diluted Sypro Orange were added. Except when indicated otherwise, maltose was present at 50 mM. Compounds tested for stability enhancement were obtained from commercial sources. Where indicated, TMAO and sarcosine were tested at 5 M in the reaction buffer, whereas 4-PBA was tested in a concentration range of 1 mM to 0.5 M. For all others, a 5 M stock was diluted to 3 M in Buffer B. Plates included a baseline control containing Sypro Orange with no protein. The pH of the final solutions did not deviate significantly from 7.
Fluorescence data were acquired on an Applied Biosciences Step-One Plus RT-PCR instrument equipped with fixed excitation wavelength (480 nm) and ROX® emission filter (610 nm). Melts were conducted from 25–95°C with a 1 °C per min increase. No difference in Tm was observed when the rate of heating was slowed to 0.5 °C per min. Above a minimum threshold of protein concentration required to observe a discrete transition, no dependence of protein concentration on Tm was observed. Data were processed using GraphPad Prism. After baseline subtraction, data were trimmed to include the boundaries and the transition of interest and normalized. The reported Tm is the inflection point of the sigmoidal curve, which was fit as described previously (34 (link)).
Fluorescence data were acquired on an Applied Biosciences Step-One Plus RT-PCR instrument equipped with fixed excitation wavelength (480 nm) and ROX® emission filter (610 nm). Melts were conducted from 25–95°C with a 1 °C per min increase. No difference in Tm was observed when the rate of heating was slowed to 0.5 °C per min. Above a minimum threshold of protein concentration required to observe a discrete transition, no dependence of protein concentration on Tm was observed. Data were processed using GraphPad Prism. After baseline subtraction, data were trimmed to include the boundaries and the transition of interest and normalized. The reported Tm is the inflection point of the sigmoidal curve, which was fit as described previously (34 (link)).
