Biophysics

Gene2Lead offers its customers the possibility to determine and optimize the stability of their targets and to detect and characterize their interactions with potential binders through three complementary technologies;

• Scanning Fluorescence (DSF); stability of the protein is determined by measuring the fluorescence of a probe whose fluorescence properties fluctuates upon binding in a hydrophobic environment. The protein is submitted to a regular increase in temperature and its denaturation (that exposes natively buried hydrophobic regions) is revealed by an increase of the fluorescence of the probe. The temperature at which the protein melts can then be calculated and is used to compare different runs. This technology is broadly used to determine optimal conditions for the study of the protein of interest, such as buffer, pH, ionic strength variations. It is also applied to the detection of partners (ions, substrates, inhibitors) that stabilize the protein and increase its melting temperature upon binding. Our DSF assays are carried on in a standard 96-well format using an iCycler (Biorad).
• Differential Static Light Scattering (DSLS); this technology (Larsson et al., 2011) is similar to the DSF in the sense that it also measures the thermal stability of the protein. But instead of using a probe, it detects the absorption of aggregates produced after denaturation. When the protein harbours natively exposed hydrophobic surfaces, DSLS is then the method of choice. As well as DSF, DSLS main application is the detection of optimal conditions and ligands. Assays are performed in 384-well plate on a Stargazer-384 (Harbringer Biotech).
• Isothermal Titration Calorimetry (ITC); An ITC experiment measures the heat produced or absorbed when a ligand binds to a protein. This method allows direct determination of the thermodynamic constants characterizing this event, such as the number of binding sites, the enthalpic and entropic variation upon binding and the Kd value. It can then be used to compare the affinities of different ligands for a same target and is, thus,  one of the major technologies in drug discovery processes. The assays are run on a ITC200 (Microcal), one of the most sensitive devices.

Gene2Leads services consists in helping our customers to design their experiment, running the experiments and providing assistance in interpreting the results.