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About Molecular Biophysics, Strasbourg, France

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Instruct Centre-FR1 offers state-of-the-art equipment and expertise to perform biophysical analysis and quality control on protein samples. Techniques include Analytical ultracentrifugation (AUC), Isothermal titration calorimetry (ITC) and Microscale Thermophoresis.

Instruments Available:

Device: Proteomelab XL-I

Device: ITC200 (MicroCal)

Virtually any molecule (small molecules, DNA, RNA, proteins, peptides, sugars, lipids, ribosomes etc) can be analysed. 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.

System components: Multi-angle light scattering detector: miniDAWN TREOS from Wyatt Technology. Equipped with the QELS module which enables measurement of the hydrodynamic radius. Differential Refractive Index (dRI) detector: Optilab T-rEX from Wyatt Technology. Chromatography system: Ettan MicroLC from GE Healthcare.

Device: Thermofluor (use of extrinsic fluorescent dye to label the proteins) and Prometheus (no dye, native intrinsic fluorescence from the proteins).

User Guide

Analytical ultracentrifugation (AUC)

AUC allows 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 state, stoichiometry, and binding constants.

Isothermal titration calorimetry (ITC) 

ITC is used to investigate all types of protein interactions, including protein-protein interactions, protein-DNA/RNA interactions and protein-small molecule interactions.

Microscale Thermophoresis (MST)

MST is a powerful new technology to quantify biomolecular interactions in a few microliter solution. MST measures changes of mobility of the molecules in microscopic temperature gradients to determine binding affinities. 

One of the binding partners is labelled using standard fluorescent labelling protocols. NanoTemper provides blue, green or red dyes optimised for protein compatibility and MST analysis. A titration series of up to 15 dilutions is prepared, where the concentration of the fluorescent binding partner is kept constant and the concentration of the unlabelled (i.e. non fluorescent) molecule is varied. 

After incubation time sufficient for the reaction to reach equilibrium (e.g. 5 minutes), the reaction is transferred into a glass capillary. The capillary is placed on the sample tray, which can accommodate up to 16 capillaries, and the sample tray is placed in the instrument. Filled capillaries are scanned consecutively and the thermophoresis signal of each sample is measured. The analysis software is used to calculate the dissociation coefficient.

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 immobilisation 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.

MST can monitor the binding of single ions (40Da) or small molecules (300Da) to a target as well as the binding of ribosomes (2.5MDa). The capillary format is easy to handle and offers maximum flexibility in the experiment scale. The capillaries come with different surface coatings to stabilise even the most complex samples in solution. Capillary volume is 5 μl. Labeling kits contain fluorescent dyes that are widely tested with MST. The labeling protocol ensures good labeling efficiency and purification. The fluorescence emission and detection fits perfect to the BLUE and RED channel in the NT.115 instruments.

MALS

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. 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

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-throughputmode to screen optimalbuffer conditions, ligands, cofactors and drugs for purified proteins. Two methods to monitor protein denaturation are available: a differential scanning fluorimetry (DSF) method and a differential static light scattering method (DSLS).

The optimisation of proteins solubility and stability properties improves the success rate of their structural studies. Changes in the thermal stability of the protein–ligand or protein-peptide complexes relative to the stability of the protein alone allow to rapidly identify promising complexes for further structural characterisation and to assign functions.

Instruct Centre

Instruct Centre FR1

IGBMC

1, rue Laurent Fries

Illkirch

Strasbourg

France

www.igbmc.fr

Molecular Biophysics, Strasbourg, France

Contacts:

Marie-Christine Poterszman
Marie-Christine Poterszman
CNRS
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Catherine Birck
Catherine Birck
IGBMC-CERBM
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Pierre Poussin-Courmontagne
Pierre Poussin-Courmontagne
IGBMC-CERBM
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