| Physical principle |
Changes in thermophoretic mobility and fluorophore microenvironment result in detectable binding induced changes in fluorescence of target molecules. |
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). |
| Readout |
Fluorescent label or intrinsic fluorescence (label-free). |
Shift in interferometric light reflection. |
Heat changes. |
| System |
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. |
| Measurement types |
Equilibrium titration, Competition titration. |
Association kinetics, Dissociation kinetics, Equilibrium titration. |
Equilibrium titration. |
| Biophysical parameters |
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. |
| Applicable molecules |
Proteins, peptides, DNA, RNA, small molecules, synthetic particles. |
Proteins, peptides, DNA, RNA. |
Proteins, peptides, DNA, RNA, small molecules. |
| Lower size limit |
100 Da. |
1 kDa. |
100 Da. |
| Upper size limit |
MDa, vesicles, synthetic particles. |
High kDa range. |
High kDa range. |
| Buffer requirements |
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. |
| Immobilization required |
No. |
Yes. |
No. |
| Labeling |
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. |
| Throughput |
Manual handling or automated high-throughput. |
96-well plate, two tips simultaneously. |
Manual handling. |
