The ISMB Biophysics Centre
at Birkbeck

Thermofluor Assay
(MyIQ RT-PCR instrument, BioRad)

Temperature denaturation of a protein gradually exposes more and more hydrophobic patches that would have been buried in the native fold. Thermofluor dye allows to monitor this as it binds to these patches and thereupon becomes much more fluorescent.

This assay can conveniently be performed in 96-well format using a real-time PCR machine. In this way, in can be used to screen the influence of buffer conditions, ligand binding or concentration on protein stability.

More accurate measurement of a melting temperature is obtained using differential scanning calorimetry or measuring the dependence of circular dichroism on temperature. However, the Thermofluor assay requires only small sample amounts and is much higher throughput. It is often used to establish suitable crystallisation conditions.

ISMB Biophysics Centre, Birkbeck, University of London
Last modified 2011