Protocol for performing and optimizing differential scanning fluorimetry experiments
Taiasean Wu, Michael Hornsby, Lawrence Zhu, Joshua C. Yu, Kevan M. Shokat, Jason E. Gestwicki
Abstract
Differential scanning fluorimetry (DSF) is a widely used technique for determining the apparent melting temperature (Tma) of a purified protein. Here, we present a protocol for performing and optimizing DSF experiments. We describe steps for designing and performing the experiment, analyzing data, and optimization. We provide benchmarks for typical Tmas and ΔTmas, standard assay conditions, and upper and lower limits of commonly altered experimental variables. We also detail common pitfalls of DSF and ways to avoid, identify, and overcome them.
Topics & Concepts
Protocol (science)Computer scienceBiological systemDifferential (mechanical device)Fluorescence spectroscopyDifferential evolutionAnalytical Chemistry (journal)ChemistryData miningChromatographyAlgorithmFluorescenceBiologyPhysicsPathologyOpticsMedicineThermodynamicsAlternative medicineProtein Structure and DynamicsEnzyme Structure and Functionthermodynamics and calorimetric analyses