Biacore生物大分子互作.ppt

What can Biacore do for you? SPR (surface plasmon resonance) technology enables real-time detection and monitoring of biomolecular events and provides quantitative information on: Specificity – how specific is the binding between two moelcules? Concentration – how much of a given molecule is present and active? Kinetics – what is the rate of association/dissociation? Affinity – how strong is the binding? Binding partners - provide identification of binding targets by linking SPR to MS What is SPR? SPR (surface plasmon resonance) arises when light is reflected under certain conditions from a conducting film at the interface between 2 media of different refractive index. In Biacore systems, the media are sample and glass of the sensor chip, and the conducting film is a thin layer of gold on the chip surface. SPR causes a reduction in the intensity of reflected light at a specific angle of reflection. This angle varies with the refractive index close to the surface on the side opposite from the reflected light (sample side). SPR response values are expressed in resonance units (RU). One RU = 0.0001o. For most proteins this is about a change in conc. Of 1pg/mm2 on the sensor surface. Biacore terminology Sensor Chips Biacore offers a range of Sensor Chips Sensor ChipsCM5 (carboxy methyl dextran) Amine coupling Amine Coupling Sensogram Thiol coupling Sensor ChipsSA (streptavidin) Sensor ChipNTA Sensor Chip NTA is designed to bind histidine-tagged molecules. Attachment of ligands via histidine-tags provides a valuable and convenient methodological interface between BIA analysis and other laboratory techniques that rely on His-tags, e.g. chelating chromatography. Attachment via a His-tag has also the advantage of orienting the ligand molecules in a homogeneous way and allowing the attachment to be carried out without significantly changing the pH or ionic strength during the coupling procedure. * * The sensorgram provides quantitative information in real-t