SPR - Instruct-CZ

SPR systems exploit the phenomenon of surface plasmon resonance to monitor the interaction between molecules in a real time. One of the interactants is immobilized on the sensor chip surface, while the other is passed over that surface in solution. Applications of SPR include biotherapeutic and drug discovery research, as well as protein activity and stability analysis. SPR is suitable also for characterization of membranes, lipids, nucleic acids and micellar systems. SPR system represents one platform for characterization of biomolecular interactions - kinetics, affinity, specificity, concentration and thermodynamics.

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SPR - Instruct-CZ

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User Guide

  • Biacore T200, SPR Imaging multichannel system exploit the phenomenon of surface plasmon resonance to monitor the interaction between molecules in a real time. One of the interactants is immobilized on the sensor chip surface, while the other is passed over that surface in solution.
  • Applications of SPR include biotherapeutic and drug discovery research, as well as protein activity and stability analysis in biopharmaceutical production. SPR is suitable also for characterization of membranes, lipids, nucleic acids and micellar systems.
  • Biacore 3000 enables measurement on up to four channels in one run. Various types of sensor chips are available - gold layer, hydrophobic layer, NTA for metalo-affinity interaction or carboxymethylated for covalent immobilization of biomolecules.
  • SPR Imaging multichannel system enables to immobilize 5 different binding partners on the sensor chip surface at the same time. 5 different ligands can be passed over these 5 different immobilized binding partners.

SPR can be used for:

  • Test of protein activity
  • Specificity - searching for binding partners, characterization of inhibitors affinity, test for cross-reactivity, eventually directly to test expression of a given protein in cell line cultures
  • Affinity (kinetics) – kinetic and equilibrium parameters of an interaction, the rates of complex formation (ka), dissociation (kd), and equilibrium association/dissociation constants can be determined.
  • Concentration determination - concentration is 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).
  • Multiple interaction during complex formation - complex formation can be monitored as each component is incorporated into a multimolecular complex.

Data collection:

  • Direct binding assay - measure the amount of analyte bound directly to the detecting molecule after sample injection
  • Binding rate measurement - monitoring of complex formation continuously as a function of time.
  • Indirect or competition (inhibition) assays - known amount of detecting molecule is mixed with sample, and the amount of free detecting molecule remaining in the mixture is measured.

Sample requirements - importance of sample preparation

  • Sample should be filtrated through 0,2 µm filter as well as a running buffer.
  • Sample environments that differ greatly from the running buffer will give rise to a bulk refractive index (RI) effect that is commonly present during an injection. Bulk refractive index effects do not affect the binding but could hide the interaction. The recommendation is that the samples should be diluted in a running buffer to minimize bulk effects or preferably to use the sample buffer as a running buffer if possible. On-line reference subtraction helps to minimize the effects of bulk.
  • 50 µl sample at least is needed for one measurement (depends on method set-up).
  • Most of the buffer compound is possible to use, 70% of ethanol and higher conc. is not allowed.
  • Immobilization of one interacting partner is essential. Choose wisely the sensor chip that will be used for immobilization of your sample. If you are in doubt, ask for expert consulting on site to minimize the risk of chip degradation.