The Biacore facility is located in Howard Hall, Rm 435, 410-706-2036.
BIAcore Technology
Quantitative measurements of binding of one ligand to another is an essential step in understanding how macromolecules interact with each other under physiological conditions. Many interactions between macromolecules are now studied in qualitative ways (e.g., yeast two-hybrid screen, gel or blot overlays, cotransfections followed by immunofluorescence or immunoprecipitation), but none of these procedures provides information about affinity or kinetics of binding. Without this information, it is difficult to be sure that the interactions one detects using such procedures actually occur in a living cell.
How Does the BIAcore System Work?
SPR DETECTION TECHNOLOGY
BIAcore biosensor technology is a versatile, highly sensitive, label-free, and easy-to-use approach to study binding interactions quantitatively, under exquisitely controlled conditions. The technology relies on the phenomenon of “surface plasmon resonance”, small changes in the reflection of monochromatic light from a metallic chip that ocur when the chip’s surface binds a protein or other molecule. The nature of the bound molecule and the “receptor” for that molecule that has been attached to the metallic surface are not critical. As a result, BIAcore technology has been used effectively to study protein-protein, protein-oligonucleotide, protein-carbohydrate, and oligonucleotide-oligonucleotide interactions. Although the binding of larger molecules is easier to study, the latest BIAcore technology (the “3000”, which we have in our Biosensor Core) can under optimal conditions reliably measure the binding of a molecule with a molecular mass as low as 360 daltons, the size of a disaccharide. The technology is therefore very sensitive.
What Can BIAcore Do?
The system is designed to measure binding in real time. Binding is initiated when a flow cell is attached to the metallic chip, prepared with a bound receptor, and a solution containing the sample of interest is introduced into the cell. The sample solution is forced over the surface of the chip along four discrete channels, and the binding to the chip surface at each of those sites is measured independently. Kinetic “on rates” are monitored in real time by an increase in surface plasmon resonance. Likewise, wash-out of the ligand results in a decrease in the signal, which also can be followed in real time to obtain the kinetic “off rate”. The same approaches can be adapted to measure maximum binding, or Bmax, making it possible to assay the absolute amounts of a macromolecule in tissues or bodily fluids. Thus, the BIAcore can provide the quantitative kinetic data needed to interpret evidence for the formation of macromolecular complexes provided by immunoprecipitations, blot overlays, or gel shift assays. Through the judicious use of the 4 different channels, replicates and controls for non-specific binding can be obtained simultaneously.
Advantages of the Technology
KINETIC DATA CURVES
The BIAcore 3000’s fluid dynamics permits accurate determinations of a wide range of “on” and “off” rates, allowing an accurate determination of binding affinity. As samples can be changed and the chip’s sensor surface regenerated rapidly and reproducibly, a wide range of binding conditions can be assayed in a short period of time. This makes it relatively easy to optimize assay conditions, which can then be applied in high through-put screens, as well as in analyses of unique biomedical samples. The volumes required for these screens are minuscule, further increasing the ability of the researcher to optimize the use of rare samples, e.g., from biopsied tissue.
Finally, the machine is highly automated, and, once accurate assay procedures have been devised, can be used to process samples robotically for many hours. This increases the efficiency of our BIAcore 3000, making it more cost effective.
Applications
The BIAcore 3000 can be used for a wide variety of laboratory procedures, just a few of which are: assaying ligand-receptor interactions (e.g., IGF-l with the IGF-1 receptor), binding of cloned fusion proteins, screening phage expression libraries, determining antigen-antibody interactions and specificity (e.g., neutralizing antibodies to viral surface glycoproteins), measurement of serum analytes (e.g., theophylline, hormones) and oncoproteins (e.g., p53 levels in serum), and studying transcription factor-DNA interactions (e.g., with the nuclear oncoprotein, ETS-1).
We have also had great success using the instrument to measure binding affinities of proteins for oligopeptide ligands, either biotinylated and bound to streptavidin-chips, or used as soluble competitive inhibitors. Of particular interest is our ability to measure binding of PDZ domains to oligopeptides of their integral membrane protein ligands.
Images are from www.biacore.com. Please see this website for more technical information.
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