Quickly characterize protein stability for construct and formulation screening Differential scanning fluorimetry (DSF) is a biophysical technique that measures protein thermal stability by tracking changes in fluorescence as a protein unfolds during a controlled temperature ramp. Using either intrinsic tryptophan fluorescence or an added dye such as SYPRO Orange, DSF reports onset of unfolding (Tonset) and melting midpoints (Tm) in little more than an hour, making it the standard first-pass assay for construct screening and formulation development. - DSF works across a wide concentration range and is applicable to most proteins, antibodies, and complex biologics — small proteins lacking aromatic residues may require an extrinsic dye rather than intrinsic fluorescence - Best suited to thermal stability ranking: construct selection, formulation screening, and comparative stability assessment - DSF reports unfolding (Tm) but not aggregation directly — pairing with static light scattering gives aggregation onset (Tagg) from the same thermal ramp Speed-up stability studies With differential scanning fluorimetry (DSF), you can quickly screen through many different proteins or formulation candidates to find the most stable ones. Instead of waiting days or weeks for an accelerated, high temp isothermal study, or even longer for a real-time stability study, DSF gets you stability answers in just a few hours by evaluating how protein conformation changes in response to a thermal ramp. Make the most of fluorescence Differential scanning fluorimetry assays work by measuring changes in fluorescence as a protein’s conformation changes. The fluorescence can be that of amino acids in the protein (the ‘intrinsic’ fluorescence of the aromatic residues, mainly tryptophan) or from an added reporter fluorophore such as SYPRO® Orange. See the change For proteins, the emitted fluorescence depends on the local environment around the fluorescent amino acids. The fluorescence spectrum of a folded protein with highly non-polar hydrophobic pockets will look different from the spectrum of an unfolded protein which has its fluorescent amino acids exposed to a more polar aqueous environment. Turn up the heat The emission spectrum of the protein will change when it melts during exposure to heat. With differential scanning fluorimetry, you are measuring changes in fluorescence as a protein unfolds in response to the increasing temperatures in a thermal ramp. Identify inflection points The onset temperature (T onset) is when proteins begin to unfold. The other inflection points (T m 1, T m 2) are the temperatures at the mid-point of unfolding events. As an added datapoint in a thermal ramp experiment, static light scattering (SLS) identifies the aggregation temperature (T agg) of the protein. Seeing T m and T agg in conjunction allows identifying which unfolding events lead to aggregation. Make the most of full-spectrum fluorescence detection Full-spectrum fluorescence is about more than just having the best signal-to-noise: it’s a key tool to figuring out when something is wrong with your DSF assay and to having a back-up plan. Small proteins might not have fluorescent amino acids, a buffer may also show fluorescence or it might quench the intrinsic protein fluorescence signal—all things that full-spectrum detection helps identify. Thankfully, differential scattering fluorimetry analysis is not limited to using the sample’s intrinsic signal. The technique can also utilize fluore
