|Binding between target and ligand results in changes of the thermophoretic mobility of the target-ligand complex due to changes in size, charge, and hydration shell.
||Binding of a ligand to a target immobilized on a fiber tip results in changes of the thickness of the molecule layer on the tip surface, which leads to changes in visible light interference patterns.
||Binding between target and ligand results in heat changes (endothermic or exothermic).
||Fluorescent label or intrinsic fluorescence (label-free).
||Shift in interferometric light reflection.
||Monolith NT.115, Monolith NT.115 pico, Monolith NT label free, Monolith NT automated by NanoTemper Technologies.
||Octet K2 by PALL FortéBio.
||Peaq-ITC by Malvern.
||Equilibrium titration, Competition titration.
||Association kinetics, Dissociation kinetics, Equilibrium titration.
||Affinity (steady-state): KD from 1 pM to 1 mM. Thermodynamics: binding enthalpy ΔH by vant-Hoff (22-45°C), derived ΔG.
||Affinity (steady-state): KD from 10 pM to 1 mM. Kinetics: association rate (kon) down to 1E-5 s-1 and dissociation rate (koff) up to 1E7 M-1s-1. Thermodynamics: binding enthalpy ΔH by vant-Hoff (25-40°C), derived ΔG.
||Affinity (steady-state): KD from 10 nM to 1 mM. Thermodynamics: directly binding enthalpy ΔH, derived free enthalpy of binding ΔG and bindin entropy ΔS. Stoichiometry: directly determinable.
|Minimum sample concentrations
||0.5 nM fluorescent target (for pico devices), 5 nM are recommended. 25 nM fluorescent target (for NT.115 devices), 100 nM are recommended. Ligand concentration dependent on affinity.
||1 μg/mL target for immobilization. Ligand concentration dependent on affinity.
||10 μM target, 100 μM ligand.
|Minimum sample volume
||50 μL target, 50 μL ligand (3 μL per titration point).
||400 μL for target immobilization, 400 μL for each ligand concentration.
||350 μL target, 60 μL ligand.
||Proteins, peptides, DNA, RNA, small molecules, synthetic particles.
||Proteins, peptides, DNA, RNA.
||Proteins, peptides, DNA, RNA, small molecules.
|Lower size limit
|Upper size limit
||MDa, vesicles, synthetic particles.
||High kDa range.
||High kDa range.
||No specific requirements, possible in complex biological liquids (serum, plasma, cell lysate, mucose fluids, urine, environmental samples). Detergents and reducing agents possible.
||No specific requirements, possible in complex biological liquids (serum, plasma, cell lysate). Detergents problematic during target immobilization.
||Buffers with low enthalpy of ionization (e.g. phosphate). Avoid reducing agents like DTT.
|Liquid handling system
||Thin glass capillaries.
||96-well plate (shaking).
||No flow system. Sample cells.
||Fluorescent label for standard MST and no label required for label-free MST. A minimum of 50 μL of a 5 μM protein stock is required for labeling.
||Different labeling options for immobilization (e.g. biotinylation; a minimum of 50 μL of a 5 μM protein stock is required. No labeling required for several immobilization strategies.
||No labeling required.
||Manual handling or automated high-throughput.
||96-well plate, two tips simultaneously.