Expertise in proteins biophysical characterization such as binding affinity, thermodynamics, structure-functions relationship studies of proteins
The Protein Biophysics Core Facility (PBCF) provides expertise, technical support, training and access to state-of-the-art instruments for the biophysical characterization of proteins, such as the production and purification of recombinant proteins, protein crystallization for structural analysis, thermodynamics analysis and molecular interactions studies. More specifically, PBCF offers services related to the sub-cloning of genes in vectors for the expression of recombinant proteins in E. coli and their purification. We also offer services related to thermodynamics analysis, methods to measure equilibrium dissociation constants (KD), using either surface plasmon resonance or isothermal titration calorimetry. The core also offers services in regard to the identification of conditions yielding diffraction quality crystals and crystal structure determination of proteins.
- Isothermal Titration Calorimetry (ITC)
- Surface Plasmon Resonance (SPR) analysis
- Differential scanning calorimeter (DSC)
- Circular Dichroism (CD)
- Data processing for binding analysis of proteins and their ligands and thermodynamics
- Sub-cloning in vectors for gene expression and overexpression of recombinant proteins in E. Coli
- Purification of recombinant proteins by Affinity, Ion-Exchange and Gel-Filtration chromatography
- Determination of conditions for obtaining protein crystals
- Crystallization robot
- Crystallization Plates Hotelling and imaging system for crystal detection remotely accessible
- Collection of X-ray diffraction patterns with X-ray generator
- Proteins structure determination and analysis from diffracting crystals
- Education
- Healthcare and social services
- Life sciences, pharmaceuticals and medical equipment
- Manufacturing and processing
Specialized labs and equipment
| Equipment | Function |
|---|---|
| New Brunswick™ Scientific Innova® 43R Incubator Shaker (2) | Refrigerated shakers specifically tailored to grow large volumes of cultures at low temperature to increase the solubility of recombinant proteins when produced in bacteria. |
| GE Healthcare AKTA Fast Purification Liquid Chromatography (FPLC) (3) | Uniquely designed to permanently connect various size exclusion columns. Columns include Superdex 75 and 200 columns to separate small (analytical) or large (preparative) quantity of proteins. The AKTAs are also equipped with ion exchange columns including Q and SP Sepharoses and Heparin columns to purify proteins based on their charges. Equipped with Superose 6 columns to separate large macromolecular complexes (up to 6 MDa). |
Malvern MicroCal VP-ITC System for Isothermal Titration Calorimetry
| ITC is the only technique that can simultaneously determine all binding parameters in a single experiment. Requiring no modification or immobilization of binding partners, ITC measures the affinity of binding partners in their native states. Measuring heat transfer during binding enables accurate determination of binding constants (KD), reaction stoichiometry (n), enthalpy (∆H) and entropy (ΔS). This provides a complete thermodynamic profile of the molecular interaction. |
| GE Healthcare Biacore X100 for Surface Plasmon Resonance (SPR) | Enables quantitative ranking of binders for a particular protein and the determination of on-rate (ka) and off-rate (koff), as well as the equilibrium dissociation constant (KD). This is particularly relevant to scientist interested in quantitatively determining the binding between any biological macromolecules. |
| Art Robbins Instruments Crystal Gryphon for protein crystallization | Crystallization robot enabling the screening, in a 96 well plate format, of diffraction quality crystals. |
| Rigaku/Formulatrix Gallery DTX and XtalDetectR UV for crystal visualisation | Protein crystal plates are stored in plate hotel (GalleryDTX) and imaged using the Desktop Minstrel UV (Xtal DetectR UV). Protein crystals can be verified by UV exposure. Data can be remotely accessed. |
| Rigaku MicroMax-007 HF X-ray generator, Rigaku R-Axis IV++ IP detector and Oxford CryoStream Cooler | Most widely used home lab X-ray source for protein crystallography. Equipped with one rotating copper anode and VariMax HR optics. PBCF’s X-ray facility is also equipped with one Rigaku R-Axis IV++ IP detector and one Oxford CryoStream Cooler as well as data processing hardware and software. |
| MicroCal VP-Capillary DSC Differential Scanning Calorimeter (DSC) | DSC directly measures the enthalpy (ΔH) and temperature (Tm) of thermally induced structural transitions in solution. This information can be used to predict shelf lives, develop purification strategies, and characterize and evaluate protein constructs or other biotherapeutic entities. In addition, it permits rapid ranking of ligand affinities to a protein target in small molecule drug discovery programs. |
Jasco J-715 Circular Dichroism (CD) Spectropolarimeter
| Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light that arise due to structural asymmetry. CD can be used to determine if a protein is folded, to characterize secondary structures (α-helix, β-sheet), for detection of changes in structure upon mutagenesis and detection of changes in the conformation of a protein upon protein:protein interaction. |