Micro Scale Thermophoresis is used to measure binding affinities in-solution while using little sample in the µL range using capillaries. The applications range from small-molecule binding events to protein-protein interactions and interactions of multi-protein complexes. MST can be performed both with labels (Monolith MST NT.115, NanoTemper) and label free (Monolith NT.LabelFree instrument, NanoTemper).
What are the sample requirements?
- Pure samples are required and concentrations determined as accurately as possible.
- The amine labelling is more critical of purity than the His tag method since all primary amines will be labelled including those on any contaminants.
- Approx. 200 µL (100 µL at least) of the labelled target is needed per one titration.
- 20-50 µL of the unlabelled ligand molecule with a concentration 20-100 fold above the expected KD per one titration.
What other specific considerations are relevant?
- MST optimized buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.05 % Tween-20 or 0.1 % Pluronic F 127. Alternatively PBS with 0.05 % Tween-20.
- For the protein labelling, commercially available labelling kits are used (amine, Cys or His-tag reactive).
- For information about Capillaries and labelling kits availability, please check with the TNA site.
- Concentration of unlabelled protein to use with a labelling kit: 100 μl of 200 nM (His-tag dye), 100 μl of 2–20 µM (amino-reactive and maleimide-reactive dyes).
- DTT and β-mercaptoethanol interfere with the labelling reaction and therefore must be avoided.
Partners offering this technique
MOSBRI reference partner site for this technique: