MicroScale Thermophoresis: A Rapid and Precise Method to Quantify Protein-Nucleic Acid Interactions in Solution

Interactions between nucleic acids and proteins are driving gene expression programs and regulating the development of organisms. The binding affinities of transcription factors to their target sites are essential parameters to reveal their binding site occupancy and function in vivo. Microscale Thermophoresis (MST) is a rapid and precise method allowing for quantitative analysis of molecular interactions in solution on a microliter scale. The technique is based on the movement of molecules in temperature gradients, which is referred to as thermophoresis, and depends on molecule size, charge, and hydration shell. Since at least one of these parameters is typically affected upon binding of a ligand, the method can be used to analyze any kind of biomolecular interaction. This section provides a detailed protocol describing the analysis of DNA-protein interactions, using the transcription factor TTF-I as a model protein that recognizes a 10 bp long sequence motif.