Affinity Selection Mass Spectrometry (AS-MS) |
- High-throughput
- Can be applied to solubilized membrane proteins
- Ligand mass detection allows verification of compound structure
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- Low-affinity binders are hard to detect
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< 10 µM |
Differential Scanning Fluorimetry (DSF) |
- Estimates the effect of the ligand on the thermal stability of a protein
- Fast and robust assay development
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- Requires a fluorescent dye
- Artefacts occur owing to fluorescence quenching or aggregation
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1 nM–100μM |
Dynamic Light Scattering (DLS) |
- Measures particle assize across the range ˜ 0.1 nm to 10 µm
- Low probe consumption
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- Low resolution
- Large particles even when present in small quantities may impact the measurement
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Translational diffusion coefficient
(Dt), Rh, dh, B22, kD, viscosity
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Fluorescence Polarization (FP) |
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- Narrow measurement window
- Sensitive to fluorescence interference
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Kd
1 nM – 1 mM
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Homogeneous Time Resolved Fluorescence (HTRF) |
- Homogenous assay
- Highly sensitive and robust
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Kd, EC50, kon, koff
1 pM – 1 mM
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Isothermal Titration Calorimetry (ITC) |
- Direct determination of thermodynamic parameters for a binding event
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- Very high protein consumption
- Requires high solubility of titrated component
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Kd, ΔH, ΔS, ΔG, stoichiometry
1 nM – 100 µM
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Microscale Thermophoresis (MST) |
- In-solution measurements
- Applicable also for challenging targets (e.g., IDPs, solubilized membrane proteins)
- Low probe consumption
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- Requires labeling of the target with a fluorophore or strong intrinsic fluorescence
- Low protein consumption
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Kd
1 pM – 1 mM
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Nano-Differential Scanning Fluorimetry (nanoDSF) |
- Estimates the effect of the ligand on the thermal stability of a protein
- Fast and robust assay development
- Relies on intrinsic fluorescence of a protein
- Low protein consumption
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- No measurements possible when protein lacks tryptophan or tyrosine residues
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Tm, Cm, &DeltaG |
Surface Plasmon Resonance (SPR) |
- Time-resolved quantification of interactions
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- Requires immobilization of the probe to the surface
- Requires highly stable protein
- Signals affected by solvent effect
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kon, koff, stoichiometry
1 pM – 500 µM
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SwitchSense |
- Molecular dynamics
- Conformational change
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- Immobilization to DNA required
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kon, koff, Kd, dh, stoichiometry
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Time-Resolved Fluorescence (TR-FRET) |
- Homogenous assay
- Highly sensitive and robust
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Kd, EC50, kon, koff
1 pM – 1 mM
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