Analytical ultracentrifugation (AUC)
AUC studies macromolecules in solution that are subjected to a centrifugal field. It combines the separation of the macromolecules and the analysis of their transportation in view of a rigorous thermodynamics. AUC is particularly suitable for studying the interactions of membrane proteins, glycosylated proteins, complexes between proteins and polymer or ARN…
It allows :
- Assessing sample homogeneity
- Determining the association state of complexes
- Providing indications on the general shape of the macromolecules
- Quantifying association constants and weak inter-molecular interactions that determine protein solubility.
- Analysing complex multi-component systems, on the basis of their density and optical properties (absorbance, refractive index and fluorescence).
BioLayer Interferometry (BLI)
BLI is an optical technique that measures variations in optical thickness at the surface of a biosensor through interference created by the reflection of a light beam on the surface of the biosensor and on the surface of the biomolecular layer. This real-time, label-free method enables the detection of biological molecules.
With the OctetRED96e, this technology facilitates the study of interactions between partners and the determination of kinetic constants of association and dissociation (kon, koff), affinity constants (KD), and the quantification of biological molecules (concentration).
The method involves a biosensor on which a ligand is immobilized; the binding of an analyte is measured on the biosensor’s surface. The response is recorded in real-time (sensorgram).
This technique is applicable to characterizing molecular interactions involving small molecules and various classes of biological macromolecules, including proteins, polysaccharides, lipids, and nucleic acids.
Fluorimeter PTI QM4
Fluorescence spectroscopy is a powerful technique that allows analysis at equilibrium or in real time, changes in the environment of intrinsic or extrinsic fluorescent probes (including tryptophan for protein, synthetic fluorophores).
Two types of quantities can be quantified with the instrument: the intensity and polarization (or anisotropy) of fluorescence, static measurements (equilibrium) or dynamic (« stopped-flow).
The technology can be applied both to the study of conformational stability of macromolecules and characterization of molecular interactions
Mass Photometer MPone
The mass photometer is an ideal tool for quality control in the protein structural analysis workflow as it can assess the molecular mass and the oligomerisation status of a sample in their native state and without the need for labels in one measurement.
Microscale Thermophoresis (MST)
Microscale Thermophoresis is a method to quantify biomolecular interactions. It measures the motion of molecules along microscopic temperature gradients and detects changes in their hydration shell, charge or size. By combining the fluorescence detection with thermophoresis, MST provides a way to measure molecular interactions.
PEAQ-ITC
The PEAQ-ITC is a highly sensitive, low volume isothermal titration calorimeter for the label-free in solution study of biomolecular interactions. It delivers direct measurement of all binding parameters in a single experiment. Requiring no modification of binding partners, either with fluorescent tags or through immobilization, ITC measures the affinity of binding partners in their native states. It works by directly measuring the heat that is either released or absorbed during a biomolecular binding event.
By measuring heat transfer during binding, ITC allows the direct determination of binding stoichiometry (n), of binding constants (KD), and variations of enthalpy (ΔH) and entropy (ΔS). Analyses at several temperatures give access to the variation of heat capacity (ΔCp) linked to the formation of a molecular complex. This provides a complete thermodynamic profile of the molecular interaction.
The enthalpy (ΔH) and the entropy (ΔS) contain information about the type of interaction and reflect the nature of the forces driving the binding.
SEC-MALS
SEC-MALS couples a size exclusion chromatography and different detectors, static light scattering, refractive index, absorbance and Dynamic light scattering or viscometer. It combines the separation of macromolecules and their characterization.
It allows:
- Determination of concentration, molecular weight and hydrodynamic radius
- Determination of aggregation and oligomerisation states: homogeneity of the sample
- Determination of conformational changes: interactions
- Determination of the stoichiometry of complexes, including for complex systems associating protein-detergent, protein-RNA, protein-polymer… It is a method often appropriated for studying interactions of membrane protein and/or glycosylated proteins.
Surface Plasmon Resonance (SPR)
SPR is an optical technique that measures changes in the refractive index on a metal surface, enabling real-time, label-free detection of biological molecules. This technology facilitates the study of interactions between partners and the determination of kinetic constants of association and dissociation (kon, koff), affinity constants (KD), and thermodynamic constants (ΔH, ΔS).
The method involves a biosensor where a ligand is immobilized; the binding of an analyte is measured during its injection through a microfluidic system with a continuous buffer flow over the biosensor surface. The response is recorded in real-time (sensorgram) and is directly proportional to the mass density of the bound biomolecules (1000 RU ≈ 1 ng/mm² ≈ 10 mg/mL).
This technique is applicable to characterizing molecular interactions involving small molecules and various classes of biological macromolecules, including proteins, polysaccharides, lipids, and nucleic acids.
