Thermofluor assay

The thermofluor assay is a simple assay based upon fluorescence. A dye emits more intensely when a protein melts over a temperature gradient as it is exposed to more and more hydrophobic residues within the core of the denatured protein. One can estimate melting temperatures, but more importantly, compare between mutants.

We can monitor fluorescence changes using a simple qPCR machine. We use the GECF 384 well plate reader.

Nonexhaustive background: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304684/

Protocol

Dyes: Thermo Protein Shift Dye (1000x) and Sypro Orange Dye (Sigma) (5000x). We use two to compare the signal. Some proteins work better with one or the other

Dye are diluted in H2O. Thermo Protein Shift Dye diluted to working stock of 5x. Sypro Orange diluted to a working stock of 25x

Proteins are diluted into buffer (keep it simple). Lysozyme is a good control and can be diluted to 10, 1 and 0.1 mg/mL.

All samples should be done in triplicate, as well as blanks for buffer.

Each well contains:

The conditions of the run were as follows (see attached)

https://wiki.epfl.ch/ptpsp/documents/SettingsforqPCR.pdf

Processing

The files are saved as .eds format and then can be opened easily on the Thermo thermal melt software on Florence's computer

The best concentration for lysozyme was 1 mg/mL. Your protein will need to be tested

The dyes made no difference in terms of Tm measurements other than that Thermo dye provided higher signal intensity.