Instrumentation
The BICF provides a wide variety of instruments for the efficient collection of biophysical data on proteins, peptides and nucleic acids.
If you are interested in being trained on, or using instruments that you are already trained on, please contact the facility manager.
Analytical Ultracentrifuge
The Beckman Optima XL-A analytical ultracentrifuge is the most versatile and accurate instrument for determining the native molecular weight and the hydrodynamic properties of a protein or other macromolecule. This technique can also be used to probe the molecular shape of proteins or other biomolecules in solution, as well as self-association or hetero-oligomeric interactions within protein samples.
Up to 3 cells and a counterbalance can be run at one time. Data can be processed using our Origin software, or by importing your files as text into non-linear regression programs such as Prism's GraphPad. Other programs such as SedFit and UltraScan might be of interest to potential users, and these are available online.
Circular Dichroism Spectropolarimeter
Secondary structural elements of a protein contribute uniquely to its circular dichroism (CD) spectrum, which can then be deconvoluted to provide net structural information. Therefore, our Jasco J-810 spectropolarimeter aids in the study of protein (and other macromolecular) conformation and stability, by providing an accurate analysis of their secondary structures using CD.
The changes of folding in protein as a function of temperature, as well as protein-ligand and nucleic acid-ligand interations can be measured by CD. Temperature is tightly controlled by a Peltier system. Cells with path lengths of 10, 1, and 0.1 mm are available. The J-810 can also be used in conjunction with our Bio-Logic SFM-400 Stopped Flow/Quenched Flow device.
Fluorometer
The Fluorolog-3 steady-state fluorometer, a sensitive, photon-counting instrument, may be used in the analysis of conformational changes in proteins through the fluorescence of endogenous tryptophan, or in a variety of fluorometric assays. The sample chamber is equipped with a stirrer, and temperature may be controlled by a circulating water bath. Basic excitation and emission scans can be easily and quickly performed to determine the peak excitation/emission wavelengths for your samples or track changes in samples after addition of ligand, and time-based acquisitions are also easily carried out. This instrument can also be used in conjunction with the Bio-Logic SFM-400 Stopped Flow/Quenched Flow device.
Isothermal Titration Calorimeter
ITC data aids in the characterization of the structure and function of macromolecules such as proteins, nucleic acids, and lipids.
The highly sensitive MicroCal VP-ITC Isothermal Titration Calorimeter can be used for the accurate determination of thermodynamic parameters of ligand binding by proteins by directly measuring the heat generated or absorbed by a protein sample upon binding of substrate. This will yield values for binding constants, reaction stoichiometry, enthalpy and entropy.
*Taken from Canton et al., JBC 2006
Differential Scanning Calorimeter
The MicroCal VP-DSC Differential Scanning Calorimeter is a highly sensitive instrument used in the thermodynamic analysis of protein denaturation. Melting temperatures, thermodynamic parameters of denaturation, and cooperativity of melting can be directly studied in this DSC, in the range of -10 to 130 degrees Celsius.
Fluorescence Lifetime Spectrometer
The PicoQuant FluoTime 200 system has single-photon timing sensitivity and contains the complete optics and electronics required for recording fluorescence decays by means of Time-Correlated Single Photon Counting (TCSPC). The system can operate at more than 80 MHz repetition rate and count rates up to several million counts per second, which allows for decay times in the picoseconds-range to be resolved. The difference in the fluorescence decay times of appropriate fluorescent dyes is a powerful method for distinguishing between molecules of interest and background or other species.
HPLC- Alliance
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The Waters Alliance HPLC system features an autosampler for repetitive automated analyses of similar samples. The instrument is equipped with a Waters 996 photodiode array (PDA) detector and is capable of producing quaternary gradients. Users typically provide their own columns, however we do have several C18 columns available for those who need them. We also have adapters now that will allow for FPLC capabilities, i.e. compatible for use with Pharmacia-style columns.
HPLC- Breeze
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The Waters Breeze system is a relatively simple HPLC system including a Waters 1525 pump for producing binary gradients, a dual wavelength detector and a Gilson FC203B fraction collector. This instrument is usually used in a "hands-on" mode for mapping or purifying peptides. Users typically provide their own columns.
Biacore X
The Biacore X is a system for real-time biomolecular interaction analysis using surface plasmon resonance technology. It is used for the measurement of kinetic and equilibrium constants, and also analysis of multiple binding patterns. Chips may be purchased through the Facility.
Stopped-Flow Module
The Bio-Logic SFM-400 Stopped Flow/Quenched Flow device may be used for rapid kinetic analyses in conjunction with either the Jasco CD or the Fluorolog-3 fluorescence instruments. It is equipped with 4 independently controlled syringes and provides submillisecond mixing with dead times down to 0.6 ms.
Dynamic Light Scattering Instrument
The Protein Solutions DynaPro Dynamic Light Scattering system can be used to study the sizes of macromolecules in the range of 0.5nm to 1um in hydrodynamic radius, and determine if the samples are monodisperse. This instrument is used to assess the suitability of protein preparations for crystallization trials.
Also from this web page:
Welcome to the BICF
The Biomolecular Interactions and Conformations Facility (BICF) is an instrumentation center within the University of Western Ontario's Department of Biochemistry in the Schulich School of Medicine & Dentistry. We provide access to state-of-the-art equipment and high quality services to researchers both on campus and beyond.
We are located in Room 394 of the Medical Sciences building on the UWO campus.
Analytical Ultracentrifugation
There are two methods of AUC that we can use:Sedimentation Equilibrium
Allows for the calculation of:
Sedimentation Velocity
Provides a rapid means for:
CD Spectropolarimetry
CD spectroscopy provides a measure of the difference between the absorption of left- and right- circularly polarized light.
For a comprehensive review, be sure to read:
