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Molecular interactions observed in a real time

Molecular interactions observed in a real time. – possibilities and limitations of SPR technology. Bogdan Walkowiak Department of Biophysics Technical University of Lodz and Department of Molecular and Medical Biophysics, Medical University of Lodz. Endpoint assay.

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Molecular interactions observed in a real time

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  1. Molecular interactions observed in a real time – possibilities and limitations of SPR technology Bogdan Walkowiak Department of Biophysics Technical University of Lodz and Department of Molecular and Medical Biophysics, Medical University of Lodz

  2. Endpoint assay

  3. Introducing a binding profile

  4. TIR – Total Internal Reflection

  5. SPR – Surface Plasmon Resonanse kx=2Pn1sin(q)/l ksp=(ng2n22/ng2+n22)0.5

  6. SPR Detection System Optical detectionunit Light source I I II II Polarizedlight Reflected light Prism II Sensor chip withgold film I Sensorgram Flow channel

  7. Microfluidic system IFC Liquid Handling

  8. Gold-Dextran Surfaces Specific layer Dextran layer • Biocompatible • Low non-specificbinding • More than 100 runs on the same surface Linker layer Gold film Glass

  9. The Sensorgram Resonance Signal (KRU) 18 Dissociation 16 Kinetics Regeneration 14 Association Concentration 12 100 200 300 400 500 600 Time (s)

  10. BiaCore sensor

  11. BiaCore sensor family

  12. Correlation between SPR response and surface concentration Signal proportional to massSame specific response for different proteins

  13. Protein concentration at the sensor surface pH 2.8 pH 4.0 pH 6.8

  14. Coupling Chemistries

  15. Protein immobilization at the sensor surface saturation concentration and immobilization activation

  16. Recombinant protein mediating fibrynolyitc process Szemraj J., Pietrucha T., Nowak P., Walkowiak B. In preparation 2002

  17. pH-dependence of MBP and DNAK interaction Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

  18. Final presentation of pH-dependence for MBP and DNAK Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

  19. Specific antibody binding to immobilized MBP protein Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

  20. Specific binding inhibition by Mab against MBP protein Selmaj K., Ćwiklińska H., Walkowiak B. In preparation 2002

  21. Acid glycoprotein binding to immobilized PAI-1 Boncela J., Papiweska I., Fijałkowska I., Walkowiak B., Cierniewski CS. J..Biol.Chem,276, 35305, 2001

  22. Vitronectin binding to immobilized PAI-1 Boncela J., Papiweska I., Fijałkowska I., Walkowiak B., Cierniewski CS. J..Biol.Chem,276, 35305, 2001

  23. Preparation of sensor surface for NCD-plasma proteins interaction Glass plate one side covered with gold film Carbon deposition by RF CVPD technology Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

  24. Raman spectrometry analysis of prepared sensor surface Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

  25. Adsorbtion of plasma proteins to the NCD surface Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

  26. Sensor for detection of platelet adhesion to immobilized fibrinogen Immobilization of rabbit anti-fibrinogen at the sensor surface Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

  27. Adhesion of blood platelets to the sensor with immobilized fibrinogen fibrinogen suspension of blood platelets regeneration adhered platelets bound fibrinogen Walkowiak B et.al. J. Wide Bandgap Materials, June 2002

  28. Cells interaction with immobilized ligands

  29. Effect of high salt concentration on antibody-ligand interaction

  30. Effect of high salt concentration on antibody-ligand interaction

  31. Sample Recovery and Fractionation Fractionate dissociating analyte for - re-injection - sequencing - mass spectrometry - electrophoresis - PCR Recover excess sample during injection - save expensive sample and reagents Recover eluted material for - enzymatic transfer assays - on-surface cleavage processes - alterations due to binding or regeneration 563

  32. BiaCore - MS

  33. Limitations Molecular weight of analyte not less than 200 Limitet chemical resistivity to organic solvents Residual interaction of analyte with dextran matrix Active for measuremet distance from a sensor surface is limited to 300 nm

  34. Thank you very much for your attention

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