Biophysical and Biochemical Platforms
Biophysics Biochemistry
Binding
MST, SPR, nanoDSF, DSF, ITC, HTRF, TR-FRET, FP, SwitchSense, Mass spectrometry, 1H and 19F NMR
STD (Saturation-Transfer Difference) NMR
nanoDSF
(differential scanning fluorometry)
MST
(MicroScale Thermophoresis)
Kinetics
SPR, HTRF, TR-FRET, SwitchSense
SwitchSense
SPR (Surface plasmon resonance)
Thermodynamics&Stoichiometry
ITC, SPR, MST &SPR, ITC, MST
Function
HTRF, TR-FRET, SwitchSense, various biochemical assays (luminescence, fluorescence, colorimetric read-out)
SwitchSense
SPR (Surface plasmon resonance)
Protein Quality
nanoDSF, DSF, DLS, Mass spectrometry
Bodipy-GTP nucleotide exchange assay
LC-MS
Complete biophysical characterization of your compound series
Comparison of methods
Advantages Disadvantages Information
obtained and
Range
Affinity Selection Mass Spectrometry (AS-MS)
  • High-throughput
  • Can be applied to solubilized membrane proteins
  • Ligand mass detection allows verification of compound structure
Low-affinity binders are hard to detect < 10 uM
Differential Scanning Fluorimetry (DSF)
  • Estimates the effect of the ligand on the thermal stability of a protein
  • Fast and robust assay development
  • Requires a fluorescent dye
  • Artefacts occur owing to fluorescence quenching or aggregation
1 nM-100uM
Dynamic Light Scattering (DLS)
  • Measures particle assize across the range ~0.1 nm to 10 um
  • Low probe consumption
  • Low resolution
  • Large particles even when present in small quantities may impact the measurement
Translational diffusion coefficient (Dt), Rh, dh, B22, kD, viscosity
Fluorescence Polarization (FP)
  • Homogenous assay
  • Narrow measurement window
  • Sensitive to fluorescence interference
Kd
1nM - 1mM
Homogeneous Time Resolved Fluorescence (HTRF)
  • Homogenous assay
  • Highly sensitive and robust
  • Requires two labels
Kd, EC50, kon, koff
1pM - 1mM
Isothermal Titration Calorimetry (ITC)
  • Direct determination of thermodynamic parameters for a binding event
  • Very high protein consumption
  • Requires high solubility of titrated component
Kd, ΔH, ΔS, ΔG,
stoichiometry
1nM - 100μM
Microscale Thermophoresis (MST)
  • In-solution measurements
  • Applicable also for challenging targets (e.g., IDPs, solubilized membrane proteins)
  • Low probe consumption
  • Requires labeling of the target with a fluorophore or strong intrinsic fluorescence
  • Low protein consumption
Kd
1pM - 1mM
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
  • No measurements possible when protein lacks tryptophan or tyrosine residues
Tm, Cm, &DeltaG
Surface Plasmon Resonance (SPR)
  • Time-resolved quantification of interactions
  • Requires immobilization of the probe to the surface
  • Requires highly stable protein
  • Signals affected by solvent effect
kon, koff,
stoichiometry
1pM - 500μM
SwitchSense
  • Molecular dynamics
  • Conformational change
  • Immobilization to DNA required
kon, koff, Kd, dh,
stoichiometry
Time-Resolved Fluorescence (TR-FRET)
  • Homogenous assay
  • Highly sensitive and robust
  • Requires two labels
Kd, EC50, kon, koff
1pM - 1mM
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