Using a fluorescently labeled protein, our DSF setup equipped with Life TechnologiesTM instruments (QuantStudio, ViiA7), and plate automation, we can efficiently screen libraries of up to 350,000 compounds and analyze hits with our in-house co-developed software (GenedataTM). Beyond hit validation purposes, we apply DSF for mode-of-action studies and can show, for instance, the importance of co-factor binding for a certain compound set. In addition, we assess the effect of different buffer conditions on protein stability. For structural biology guidance, we evaluate the folding behavior of different protein constructs. Furthermore, we use DSF for fragment screens, which can result in the generation of a highly valuable tool compound.
For thermal shift assays with membrane proteins, common dyes are not compatible due to their non-specific hydrophobic interactions. Instead, we use a thiol-reactive fluorescent dye, CPM (7-Diethylamino-3-(4'-Maleimidylphenyl)-4-Methylcoumarin). Using a CPM assay, we can successfully study substrate-induced thermal stabilization of GPCRs and SLC transporters.
With nano-differential scanning fluorimetry (nanoDSF), NUVISAN has another label-free method in place for measuring protein stability. Like DSF, the effect of buffers, co-factors, and ligands on protein stability can be evaluated with high levels of sensitivity. The throughput of nanoDSF is lower than for DSF, but it is one of our preferred methods to guide the optimization of protein purification at all steps, including production of membrane proteins, due to its low protein requirements.
