BIAcore (Biomolecular interaction analysis): principles by HC12042103828

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									               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
     –   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
         (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 K D 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
               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




          Inhibiting Ras/Gef interaction with
 450
 400
                   LMW compounds
 350
 300
RU
 250
 200
 150
 100
 50                     Unpublished (in collaboration with F Peri)
     0
         -50   0   50   100   150    200    250    300    350
                                                                     Gobbi M et al, J Biol Chem (2006) 281:843-9
                              Time (sec)

								
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