Molecular Biotechnology | BIAcore 3000
Surface Plasmon Resonance (SPR) has been used to monitor macromolecular interactions in real time. The BIAcore 3000 system is an instrument that uses SPR technology for measuring the interactions of macromolecules with each other, and with small ligands. One of the ligands is immobilized on carboxymethylated dextran over a gold surface, while the second partner (analyte) is captured as it flows over the immobilized ligand surface. Most ligands can be directly immobilized onto the surface of the chip via amino groups, carbohydrate moieties, or sulfhydryl groups. Others are immobilized indirectly through the use of biotinylation of the ligand (such as biotinylated peptides or oligonucleotides), or through immobilized monoclonal antibodies (such as anti-GST). Typical amounts of a protein ligand needed for an immobilization reaction is about 1 µg. The immobilized ligands are remarkably resilient and maintain their biological activity. The bound analytes can be stripped from the immobilized ligand without affecting its activity to allow many cycles of binding and regeneration on the same immobilized surface. Interaction is detected via SPR, in real time, at high sensitivity, without the use of radioactivity. Affinity is measurable in the range of 105 – 5×1011M-1. Because the same affinity may reflect different on-rates and off-rates, this instrument excels over most other methods of affinity measurements in that it measures on-rates (ka in the range of 103 – 5×106 M-1s-1) and off-rates (kdof 10-5 – 10-2 s-1). Concentration determination experiments are also feasible.
Biacore can be used for measuring the binding constants of macromolecular interactions (protein-protein, nucleic acids – protein, protein-ligand etc) and in many other applications such as, epitope mapping, ranking antibody affinities, concentration measurements, characterization of mutant proteins and Ligand fishing.
The instrument and services are available on a fee-for-service basis to Cleveland Clinic researchers as well as