Comparison between MST, BLI, and ITC

	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): K_D from 1 pM to 1 mM. Thermodynamics: binding enthalpy ΔH by vant-Hoff (22-45°C), derived ΔG.	Affinity (steady-state): K_D from 10 pM to 1 mM. Kinetics: association rate (k_on) down to 1E-5 s^-1 and dissociation rate (k_off) up to 1E7 M^-1s^-1. Thermodynamics: binding enthalpy ΔH by vant-Hoff (25-40°C), derived ΔG.	Affinity (steady-state): K_D 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.