The Protein Biophysics Core Facility (PBCF) is located on the 4th floor in Roger Guindon Hall, at the Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, University of Ottawa. The PBCF houses a wide range of instrumentations suitable for the biophysical characterization of proteins. PBCF is equipped with facilities needed for the production and purification of recombinant proteins, for protein crystallization as well as for conducting binding studies. PBCF offers services related to the sub-cloning of genes in vectors for the expression of proteins in bacteria as well as services related to the overexpression and purification of recombinant proteins in E. coli. We offer services related to the determination of equilibrium dissociation constants (KD) for a protein to its ligand using surface plasmon resonance or isothermal titration calorimetry. Finally, the core also offers services in regard to the identification of conditions yielding diffraction quality crystals and crystal structure determination of proteins.
Two refrigerated shakers/incubators Innova 43R for protein overexpression (New Brunswick Scientific)
These shakers are specifically tailored to grow large volumes of cultures at low temperature to increase the solubility of recombinant proteins when produced in bacteria.
Two AKTA Fast Purification Liquid Chromatography (FPLC) (GE Healthcare)
These FPLCs are uniquely designed in such a way that various size exclusion columns are permanently connected to these systems. These 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. Finally, these systems are equipped with Superose 6 columns to separate large macromolecular complexes (up to 6 MDa).
MicroCal VP-ITC for Isothermal Titration calorimetry (Malvern)
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.
Biacore X100 for Surface Plasmon Resonance (SPR) (GE Healthcare) (CURRENTLY OUT OF SERVICE)
The SPR enables quantitative ranking of binders for a particular protein. This instrument also enables 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.
Crystal Gryphon for protein crystallisation (Art Robbins Instruments) (CURRENTLY OUT OF SERVICE)
The Crystal Gryphon is a crystallization robot enabling the screening, in a 96 well plate format, of diffraction quality crystals.
Gallery DTX and XtalDetectR UV for crystal visualisation (Rigaku/Formulatrix)
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-007HF x-ray generator and Rigaku R-Axis IV++ IP detector (Rigaku)
Rigaku MicroMax-007HF is the most widely used home lab X-ray source for protein crystallography. It is 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 Cryocooler as well as data processing hardware and software.
MicroCal VP Capillary DSC Differential scanning calorimeter (DSC) (CURRENTLY OUT OF SERVICE)
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 spectrometer for Circular Dichroism (CD) (Jasco)
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.
Services and Fees
Protein Expression and Purification*
|Protein expression and solubility||$40/screen|
|AKTA Fast Purification Liquid Chromatography||$12/hr|
|Surface Plasmon Resonance (CURRENTLY OUT OF SERVICE)||$10/hr|
|Isothermal Titration Calorimetry||$10/hr|
|Differential Scanning Calorimetry (CURRENTLY OUT OF SERVICE)||$10/hr|
|Circular Dichroism Spectroscopy||$10/hr|
|Gryphon- 96 well crystallization plate (CURRENTLY OUT OF SERVICE)||$40/plate|
|24 well crystallization plate||$30/plate|
|Crystal Screening and Data Acquisition||$25/hr|
Training and Technical Support
A 10% incentive fee is added to the price for the consumables used for preparing and filtrating buffers to purify proteins