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Utilizzo della tecnologie BIacore nello studio delle interazioni proteina-proteina e proteina ligando Marco VANONI DIPARTIMENTO DI BIOTECNOLOGIE E BIOSCIENZE.

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Presentation on theme: "Utilizzo della tecnologie BIacore nello studio delle interazioni proteina-proteina e proteina ligando Marco VANONI DIPARTIMENTO DI BIOTECNOLOGIE E BIOSCIENZE."— Presentation transcript:

1 Utilizzo della tecnologie BIacore nello studio delle interazioni proteina-proteina e proteina ligando Marco VANONI DIPARTIMENTO DI BIOTECNOLOGIE E BIOSCIENZE

2 BIAcore (Biomolecular interaction analysis): principles

3 BIAcore : applications Specificity –The extent to which different molecules interact with a single partner immobilized on a sensor surface reveals the specificity of an interaction Qualitative (Yes/No) answers –Search for binding partners –Screen for inhibitor specificity –Test for cross-reactivity –Look for activity after purification Kinetics and affinity determination –The kinetics of an interaction, i.e. the rates of complex formation (k on ) and dissociation (k off ) can be determined from the information present in a sensorgram, by fitting the data to interaction models. –For a simple 1:1 interaction, the equilibrium constant K D is the ratio of the kinetic rate coinstants, k off /k on Concentration –Determined by monitoring the interaction of a molecule with a prepared sensor surface in the presence of a target molecule in solution (solution inhibition) or excess analyte (surface competition). Concentrations are calculated by interpolation of the binding responses on a calibration curve Multiple interactions during complex formation –Complex formation can be monitored as each component is incorporated into a multimolecular complex

4 BIAcore (Biomolecular interaction analysis): examples Phosphorylation-dependent binding of a Cki to a cyc/Cdk complex Barberis M et al, BBRC (2005) 336: RU Time (sec) Inhibiting Ras/Gef interaction with LMW compounds Unpublished (in collaboration with F Peri) Probing prion fibril formation Gobbi M et al, J Biol Chem (2006) 281:843-9


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