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Utilizzo della tecnologie BIacore nello studio delle interazioni proteina-proteina e proteina ligando

Marco VANONI. Utilizzo della tecnologie BIacore nello studio delle interazioni proteina-proteina e proteina ligando. DIPARTIMENTO DI BIOTECNOLOGIE E BIOSCIENZE. BIAcore (Biomolecular interaction analysis): principles. BIAcore : applications. Specificity

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Utilizzo della tecnologie BIacore nello studio delle interazioni proteina-proteina e proteina ligando

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  1. Marco VANONI Utilizzo della tecnologie BIacore nello studio delle interazioni proteina-proteina e proteina ligando 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 (kon) and dissociation (koff) 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 KD is the ratio of the kinetic rate coinstants, koff/kon • 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. Phosphorylation-dependent binding of a Cki to a cyc/Cdk complex Probing prion fibril formation 450 400 350 300 RU 250 200 150 100 50 Barberis M et al, BBRC (2005) 336:1040-8 0 -50 0 50 100 150 200 250 300 350 Time (sec) Inhibiting Ras/Gef interaction with LMW compounds Unpublished (in collaboration with F Peri) Gobbi M et al, J Biol Chem (2006) 281:843-9 BIAcore (Biomolecular interaction analysis): examples

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