Differential scanning fluorimetry

Differential scanning fluorimetry follows fluorescence while a protein sample undergoes controlled heating. Changes associated with protein unfolding provide a transition curve and an apparent protein unfolding midpoint. Comparing matched conditions turns the method into a thermal shift assay.^[ref]

In a common implementation, an environment-sensitive fluorescent dye reports changes as normally buried regions become exposed. Compare otherwise matched protein-plus-dye samples with and without a candidate ligand, rather than comparing measurements made with different buffers or heating programs. Fluorescent compound-only and dye-only controls can help diagnose reporter interference; these are proposed controls, not specific settings claimed for every published assay.

For an illustrative result, midpoints of $320\mathrm{K}$ and $324\mathrm{K}$ give a thermal shift of $+4\mathrm{K}$. The measured quantity is a change in a heating-dependent transition. An independent ligand binding or activity measurement is needed to assess the proposed molecular mechanism, as illustrated by the comparisons with isothermal titration calorimetry in the 2004 evaluation.^[ref]