Table 2. Troubleshooting table.

Step	Problem	Possible reason	Solution
1	Insolubility of dye in buffer	Protein buffers are incompatible with dye	Addition of DMSO up to a final concentration of 5–10% (vol/vol) in the assay may be required to keep dye soluble
3	Small quantity of protein to test	Low-yielding membrane protein; smaller- scale reactions	Use a lower concentration of protein (we used 10 μM in 40 μl as shown in Supplementary Fig. 2); adjust controls accordingly
6	Slow reaction with DCIA	Buried cysteine mutants within the transmembrane region of MscS	Extend the cycle time to 2 min to allow sufficient time for DCIA to react with cysteines
7	Low fluorescence signal	Protein precipitation; thermostable protein	Addition of 0.05% DDM (wt/vol) detergent to aid in unfolding of protein and eliminate protein precipitation
8	Decrease in fluorescence signal at high temperatures	Protein aggregation12 and photobleaching	Interpret data at an appropriate temperature before protein aggregation has occurred. In most cases, the thermofluorescence profile reaches a characteristic plateau after the highest fluorescence signal has been observed