Label-Free Interaction Analysis with Microscale Thermophoresis

Abstract

The analysis of bio-molecular interactions, such as protein-protein or protein-nucleic acids provides novel insights into basic cellular processes. Techniques currently employed to interrogate these interactions typically require some type of labeling by fluorescent or radioactive tags or rely on a surface-coupling of one binding partner (SPR).The attachment of a binding partner to either a tag or to a surface may alter or inhibit the functionality of the binding characteristics. So far, truly label-free interaction studies are mostly performed with isothermal titration calorimetry (ITC).Here we show a new label-free and preparation-free technique to analyze the affinity of bio-molecular interactions based on the recently invented method of microscale thermophoresis (MST). The technique allows high fidelity measurements of protein-DNA interactions, the binding of receptors to their ligands and of low molecular weight binders to target proteins.Label-free MST uses the directed movement of molecules in optically generated microscopic temperature gradients. This thermophoretic movement is determined by the entropy of the hydration shell around the molecules. Almost all interactions between molecules and also virtually any biochemical process relating to a change in size, stability and conformation of molecules alter this hydration shell and can thus be determined and quantified. Up to now, reports on this technique rely on a fluorescent tag added to one of the binding partners. Here, we show the use of the intrinsic tryptophane fluorescence. Most proteins possess one or few tryptophane residues, offering a widespread use of this source of fluorescence.We exemplify the technique by investigating the binding of ligands to membrane receptors, quantifying protein-DNA interactions, ion-protein interactions and screen for small molecule binders to the kinase p38. All groups of interactions were readily accessible by label-free MST and the measured affinities confirmed values reported in the literature.