Background

Protein-protein interactions are often characterised in terms of the associated dissociation constant, K_D. The binding constant can be established using a variety of techniques, such as isothermal calorimetry (ITC), NMR spectroscopy, and surface plasmon resonance (SPR). An alternative method is based on thermophoresis, a phenomenon where distinct molecules (such as a protein-protein complex versus individual proteins) respond differently to a temperature gradient (Duhr and Braun, 2006; Seidel et al., 2013). This method is rapid, no sample immobilisation is needed and the sample requirement is low. Briefly, one of the proteins is labelled with a fluorescent dye and kept at a constant, low concentration. A dilution series is set up, where the other protein is diluted up to 16 times, creating a vast concentration range. The two proteins are subsequently mixed and loaded into capillaries, which are scanned in the Monolith^TM NT.115 instrument, developed and sold exclusively by NanoTemper. The samples are subjected to a temperature gradient, and the movement of the fluorescently labelled molecule is tracked. The difference in the fluorescence of the molecule at the initial temperature and at the new temperature is used to generate a binding curve as a function of the concentration of the unlabelled protein.