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Biophysical Characterisation

 

Protein Stability

  • 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.

Protein Multimerisation and Complex Formation

  • 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.

Macromolecular Interaction - ITC

  • 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.

 

Macromolecular Interaction - SPR

  • The 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.

NKI Protein Facility                                                                                                                                                                                                         Last updated: 16 April 2012

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Plesmanlaan 121

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