MST
Microscale Thermophoresis (MST)
MST measures binding by detecting changes in the directed movement of molecules along a microscopic temperature gradient created by an infrared laser. A fluorescently labeled binding partner changes its thermophoretic behavior (and often fluorescence) when bound, allowing dissociation constants to be measured in free solution across a very wide affinity range.
Instrument
NanoTemper Monolith NT.115
Kd range 1 nM - 500 mM
Sample Requirements
- Standard binding (Kd) measurement: One fluorescently labelled binding partner held at low, constant concentration (often 1 – 50 nM); the unlabelled partner titrated across ~16 serial dilutions.
- Labelling options: Amino-terminal or lysine dye conjugation, His-tag labelling, SNAP-tag labelling or Cy5-labeled ligands.
- Label-free mode: Uses intrinsic Trp fluorescence; requires higher protein concentration and a suitable instrument.
- Suitable sample types: Proteins, peptides, nucleic acids, lipids, chemical compounds, ions, liposomes, and crude-lysate targets are all feasible.
Experiment Examples
- Measuring the affinity of protein–protein interaction (e.g. antibody-antigen) in solution.
- Quantifying protein-DNA/RNA interaction using a fluorescently labelled oligonucleotide.
- Determining small-molecule or fragment affinity to a labelled target protein during early drug discovery.
- Running a competition assay to measure the IC50 of an inhibitor displacing a known binder.
- Measuring cofactor, lipid, metal ion, or peptide binding to a protein.
- Comparative Kd measurements with protein/ligand variants (e.g. mutants).
