Transcript BIAcore (Biomolecular interaction analysis): principles

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
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Specificity
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Qualitative (Yes/No) answers
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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
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Search for binding partners
Screen for inhibitor specificity
Test for cross-reactivity
Look for activity after purification
Kinetics and affinity determination
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The extent to which different molecules interact with a single partner immobilized on a
sensor surface reveals the specificity of an interaction
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
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Complex formation can be monitored as each component is incorporated into a
multimolecular complex
BIAcore (Biomolecular interaction analysis): examples
Phosphorylation-dependent binding
of a Cki to a cyc/Cdk complex
Probing prion fibril formation
Barberis M et al,
BBRC (2005) 336:1040-8
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Inhibiting Ras/Gef interaction with
LMW compounds
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RU
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Unpublished (in collaboration with F Peri)
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Time (sec)
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Gobbi M et al, J Biol Chem (2006) 281:843-9