Purpose
Thermal stability (Tm), label-free, from intrinsic tryptophan/tyrosine fluorescence.
Setup
- Standard-grade capillaries, ~10 µL of ~0.5-1 mg/mL protein in its SEC/storage buffer (PBS pH 7.4)
- Ramp 20→95 °C at 1 °C/min
- Read the 350 nm/330 nm fluorescence ratio continuously; Tm is the inflection point of that ratio (first-derivative peak), not the raw ratio value itself
- Run in duplicate at minimum; flag >1 °C spread between replicates
Bonus read
The same run's back-reflection/scattering channel gives an aggregation-onset temperature (Tagg) alongside Tm — worth recording even though it isn't a required field here, since a low Tagg well below Tm is an early warning for the same surface-hydrophobicity problem Protocol/SEC would catch later.