The Protein Facility of the Netherlands Cancer Institute (NKI) in Amsterdam has a strong record in structural biology using macromolecular X-ray crystallography, Electron Microscopy and sample preparation, and is also extremely well-equipped for studying molecular biophysics.
With respect to Molecular Biophysics we offer to our users fluorescence-based fast kinetics and high-throughput experiments, ITC, MST, MALS, SPR and Thermal Shift Assays.
Interaction between macromolecules can be studied using isothermal titration calorimetry (ITC). A ligand is titrated into a macromolecular solution and the change in heat caused by the interaction is measured. From the resulting binding curve, the association constant (Ka; is 1/dissociation constant Kd), the binding enthalpy (DH), entropy (DS) and the stoichiometry can be determined. The advantage of this technique is that binding is measured in solution without immobilization; disadvantage is the rather large amount (0.2 - 1.5 mg) of macromolecule that is required for a single experiment. Affinities (Kd values) in the nanomolar and low micromolar range can be determined.
A multi-angle static light scattering (MALLS) device is available to determine an accurate molecular weight of purified proteins in solution. This can be useful to assess protein homogeneity, multimerisation and protein complex formation. The device is coupled to a gelfiltration column such that each peak that elutes from the column is analyzed for its composition (in terms of molecular weight). About 0.5 mg of purified protein is required for one experiment.
The NKI protein facility has access to a BIACORE T100 for studying biomolecular interactions by surface plasmon resonance (SPR). Macromolecule of interest is immobilized on a chip and a solution containing the binding partner is flown over the chip. Binding is measured as the change in mass bound to the chip and from the binding curves the association- and dissociation rate constants (ka and kd respectively) and the dissociation constant (Kd) are calculated. Relatively small amounts of protein (in the order of micrograms) are required for the binding studies.
The thermal stability of purified proteins can be examined using the thermofluor assay: The temperature of a protein solution containing a fluorescent probe is increased in a RT-PCR machine and protein unfolding is monitored by the change in fluorescence due to binding of the probe to hydrophobic ('unfolded') protein sites. From the curve, an apparent melting temperature can be calculated. The experiment is performed in 96-well format and can be used to identify the most suitable storage buffer and/or can be helpful in identifying promising crystallisation conditions.
Prometheus measures thermal stability by monitoring internal fluorescence and aggregation by monitoring back-scattering