Biophysical assay services

GCI is a technique based on measuring refractive index changes within an evanescent field near a sensor surface. Following target immobilization to the surface, the in-solution molecules (known as the analytes) which interact with that target, cause a small intensification of the local refractive index. GCI technology can monitor these refractive changes over time and can therefore provide both kinetic rates and affinity constants of the analyte-target binding. In addition, extremely low concentrations of the analyte can be measured to provide accurate measurements of the amount of active analyte in the samples. However, in contrast to other optical methods, the light in GCI can travel through the entire length of the sample resulting in more binding events to contribute to the overall signal. It also has the advantage of being able to monitor very low abundance interacting analytes in crude samples such as biofluids due to its non-clog system.

Using Creoptix® WAVEsystem

Used for: library screening (including fragment libraries), orthogonal hit verification, hit validation, SAR, affinity profiling (KD) and kinetic profiling (ka, kd, residence time).

ITC is a biophysical method that measures the interaction of two binding molecules in-solution. Not only can it measure accurate affinities but can also be used to determine the thermodynamic parameters (enthalpy ΔH and entropy ΔS). It is used to study the binding of small molecules, peptides or nucleotides to proteins and other macromolecules. The instrumentation consists of two cells, one containing the macromolecule and the other containing buffer which acts as the reference cell. The small molecules to be studied are placed in the syringe and are injected into the sample cell. Minute heat changes that occur upon interaction are monitored and the resulting trace can be plotted to provide affinity, thermodynamics and accurate stoichiometry. ITC is considered the gold standard technique in characterizing binding affinity and thermodynamics of ligands for proteins.

Using a MicroCal PEAQ-ITC

Used for: protein QC, validation of other biophysical techniques, orthogonal hit verification, hit validation, SAR, affinity profiling (KD) and thermodynamic profiling (ΔG, ΔH, ΔS)

DSF utilizes specialized fluorogenic dyes to measure changes in the thermal denaturation temperature of a protein under varying conditions such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mutation. The dyes bind non-specifically to hydrophobic surfaces, and water strongly quenches its fluorescence so that 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) is obtained by gradually increasing the temperature to unfold the protein and measuring the fluorescence at each point. Curves are measured for protein only and protein + ligand, and ΔTm is calculated. DSF is commonly used in the early stages of drug discovery to screen thousands of ligands for binding against the target protein.

Using RT-PCR instruments

Used for: library screening (including fragment libraries), orthogonal hit verification, hit validation, SAR and affinity profiling (KD)

In SPR assays, target molecules, most frequently proteins, are immobilized on a prepared gold sensor surface and a sample containing a potential interacting partner in solution is injected over the surface through a series of flow cells. During the interaction, polarized light is directed toward the sensor surface and the angle of minimum intensity reflected light is detected which changes as molecules bind and then dissociate. The SPR instrument then produces a sensorgram which is a real-time record of this interaction. SPR allows real-time, label-free detection of biomolecular interactions and is used primarily in pharmaceutical development, quality control for assay development, and basic life science research.

Using either a Biacore T200 or a Biacore 8k

Used for: library screening (including fragment libraries), orthogonal hit verification, hit validation, SAR, affinity profiling (Kd) and kinetic profiling (ka, kd, residence time)

MST is a technology for the analysis of interactions between biomolecules. It is based on the detection of a temperature-induced change in fluorescence of a target as a function of the concentration of a non-fluorescent ligand. Two effects take place during the heating of the sample by the laser. Firstly, temperature-related intensity change (TRIC) of the fluorescent probe, which can be affected by binding events, and secondly on thermophoresis, the directed movement of particles in a microscopic temperature gradient. When a temperature gradient is applied to the sample, MST can detect very small changes in the microenvironment of the fluorescent probe, as well as changes in the hydration shell of biomolecules. These changes alter the detected fluorescence and can be used to determine binding affinities. Similar to ITC, MST measures interactions directly in solution without the need for immobilization to a surface.

Using a NanoTemper NT115pico
Used for: protein QC, validation of other biophysical techniques, library screening (including fragment libraries), orthogonal hit verification, hit validation, SAR and affinity profiling (KD)