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).
Circular Dichroism (CD)
Studying macromolecules (and in particular proteins) by circular dichroism allows to obtain information about their folding. Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to structural asymmetry. For proteins, far UV (180-260 nm) and near UV (250-330 nm) circular dichroism measurements give insight respectively into their secondary structure content and their tertiary organization.
Dynamic Light Scattering (DLS)
DLS is used to measure particle size and molecule size. This technique measures the diffusion of particles moving under Brownian motion, and converts this to size and a size distribution using the Stokes-Einstein relationship.
It allows to:
- Characterise the size/monodispersity of various particles including proteins and micelles
- Determination of molecular sizes (diameter 0.3nm to 10.0 microns)
- Detection of aggregates / oligomerisation
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.
Nanoparticle Tracking Analysis (NTA)
The NanoSight instrument utilizes Nanoparticle Tracking Analysis (NTA) to characterize nanoparticles from 10nm -2000nm in solution. This technology utilizes the properties of both light scattering and Brownian motion in order to obtain the size distribution and concentration measurement of particles in liquid suspension. A fluorescence mode provides differentiation of labelled particles.
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.
Thermal Shift Assay (TSA): denaturation
This q-PCR machine, combined with SYPRO orange dye, is used to mesure denaturation / stability of protein with temperature.
SYPRO Orange binds nonspecifically to hydrophobic surfaces, and water strongly quenches its fluorescence. When the protein unfolds, the exposed hydrophobic surfaces bind the dye, resulting in an increase in fluorescence by excluding water.
The stability curve and its midpoint value (melting temperature, Tm also known as the temperature of hydrophobic exposure, Th) are obtained by gradually increasing the temperature to unfold the protein and measuring the fluorescence at each point.