Protocol 1 - Ligand binding

Immobilization is a procedure to attach ligand on the SPR sensor surface in order to facilitate an interaction measurement. Usually ligands are covalently attached by reactive group but they can be also captured e.g. with streptavidin. Molecular properties such as molecular weight, reactive group (-NH2, -SH, -COOH) and purity affect the choice of appropriate immobilization technique. Aminocoupling is mostly used covalent immobilization technique due to the fact that most macromolecules contain several amine groups.

Typical immobilization protocol using aminocoupling:

  1. Set the temperature and prime the system
  2. Insert carboxymethyldextran (CMD) sensor slide in the MP-SPR Navi instrument
  3. Start buffer flow
  4. Clean the sensor surface injecting cleaning solution.
  5. Activate the surface injecting NHS/ EDC mixture to the channels.
  6. Inject protein one of the channels. Leave one channel without protein to be used as reference channel.
  7. Deactivate the surface injecting ethanolamine to the channels.
  8. Check proper immobilization level

Pre-concentration can be done before surface activation to ensure proper immobilization conditions for particular ligand used.

Protocol 2 - Analyte interaction

After creating a sensor surface by immobilizing a protein, the analyte interaction can be measured. A typical protocol for the interaction measurement is as follows:

  1. Set the temperature
  2. Start buffer flow
  3. Prepare series of analyte concentrations
  4. Inject middle analyte concentration
  5. Inject regeneration solution to remove analyte from the ligand if dissociation is slow.
  6. Inject analyte samples from lowest to higher concentration. Recommended is 5 concentrations (minimum 2).
  7. Measure another analyte interaction or stop the measurement and clean the instrument
  8. Analyze data with MP-SPR NaviTM DataViewer and TraceDrawer for MP-SPR NaviTM to determine affinity and kinetic values of the interaction.

For further information: