Instruct centre - France 1 offers a large panel of state-of-the-art equipment and expertise in biochemistry and biophysics to perform quality control and functional analysis on protein samples.
Analytical ultracentrifugation (AUC) is a powerful technique for the characterisation of macromolecules and macromolecular self- and hetero-association processes in solution. Two types of complementary experiments, sedimentation velocity and sedimentation equilibrium, can be performed in an analytical ultracentrifuge which is a high-speed centrifuge equipped with an optical detection system. The observation of macromolecules or macromolecular complexes sedimentation gives access to their hydrodynamic and thermodynamic properties, including their size, shape, molar mass, degree of heterogeneity, oligomeric
At IGBMC, the Isothermal titration calorimetry (ITC) is used to investigate all types of protein interactions, including protein-protein interactions, protein- DNA/RNA interactions and protein-small molecule interactions.
Device: ITC200 (MicroCal)
Measure changes of mobility of the molecules in microscopic temperature gradients to determine binding affinities.
Microscale Thermophoresis (MST) is a powerful new technology to quantify
biomolecular interactions in a few microliter solution. The MST method
is based on thermophoresis, the directed movement of molecules in a
temperature gradient, which strongly depends on a variety of molecular
properties such as size, charge, hydration shell or conformation.
Virtually any molecule (small molecules, DNA, RNA, proteins, peptides,
sugars, lipids, ribosomes etc) can be analyzed. The thermophoresis is
detected and quantified using either covalently attached or intrinsic
fluorophores. For example, the thermophoresis of a protein typically
differs significantly from the thermophoresis of a protein–ligand
complex due to binding-induced changes in size, charge and solvation
energy. For deriving binding constants, multiple capillaries with
constant concentrations of protein and increasing concentration of
ligand are scanned consecutively and thermophoresis is detected. The
analysis software is used to plot and fit the change in thermophoresis
to yield a Kd. This technology has several advantages over other
standard techniques to analyze interactions, such as surface plasmon
resonance (SPR) and isothermal microcalorimetry (ITC). It can measure
affinities in free solution without surface immobilization with low
sample consumption and within sub-nM to mM range. Experiments can be
carried out with a broad range of solution conditions, including
detergent mixtures and complex bioliquids.
Size exclusion chromatography (SEC) coupled with multiangle light scattering (MALS) is a straightforward technique to determine the accurate molar mass and the average size of proteins and macromolecular complexes in solution. Indeed, MALS can measure the absolute molar mass and size of molecules without the use of reference standards.
One of the major application is the determination of the size and stoichiometry of tightly bound heterocomplexes, such as protein/protein, protein/DNA, protein/RNA and protein/detergent interactions.
Thermal shift assay is a thermodenaturation assay to monitor the thermal stability of proteins and investigate factors affecting this stability. This rapid and simple technique is used in high-throughput mode to screen optimal buffer conditions, ligands, cofactors and drugs for purified proteins.