Protein Purification-free Method of Binding Affinity Determination by MicroScale Thermophoresis:
MicroScale Thermophoresis is a versatile and powerful technique which allows you to perform MST measurements in cell lysate without the need to purify the protein. In this video publication, the group of Nadya Tarasova introduces you to the technology of MicroScale Thermophoresis and provides a step-by-step guide how to perform MST measurements in cell lysate using a GFP fusion protein. All important steps are presented including preparation of the cell lysate, assay setup, MST measurements at the Monolith NT.115 and MST data analysis.
Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of the currently available methods of Kd determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for the determination of binding affinity by MicroScale Thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.
MST studies of non-purified GFP-fused proteins were found to be very convenient for quantitation of binding affinities for many proteins and ligand types. There is no doubt that wider use of the method in many laboratories will allow further broadening the applications of MST-based protein binding studies.