Technology comparison
MST | BLI | ITC | |
---|---|---|---|
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. |