The BIA Core 3000

1. The BIACore 3000 Theory, Applications & Techniques.

3. How does it work? -Solid Phase ligand binding -Surface Plasmon Resonance What can it do? -Realtime Binding Kinetics. -Orphan Receptor Studies. What can you do?-Data Analysis -Some Examples so far... Topics:

4. Basic Principle • A binding molecule is bound to the sensor surface.(eg a peptide) • Another (the analyte) is passed over the surface and binds to it.

5. Direct coupling of Ligand to Surface. • Indirect, via a capture molecule (eg a specific IgG). • Membrane anchoring, where the interacting ligand is on the surface of a captured liposome. Study Methods

6. Sensor Chips: CM5 SA NTA HPA Pioneer Chips

7. Sensor Chip CM-5:Carboxymethylated dextran coated surface. Allows covalent coupling via -NH2, -SH, -CHO & -COOH groups:

8. Sensor Chip SA :Streptavidin coated dextran surface. • Capture biotinylated DNA, proteins, lipids etc.

9. Sensor Chip NTA: Bind His-tagged ligands to chelated nickel

10. Sensor Chip HPA: Anchor membrane-bound ligands on a hydrophobic surface

11. Pioneer Chips • Pioneer Chip L1 (99-1000-05): Lipophilic Surface • Facilitates the formation of lipid bilayers

12. Pioneer Chips: • Pioneer Chip C1 (99-1000-04): Flat carboxymethylated surface. • Useful for work with bulky components, such as cells and virus particles • Pioneer Chip B1 (99-1000-02): Low degree of carboxylation • Reduces non-specific binding of molecules which have a high positive charge, eg cell culture supernatants.

13. Pioneer Chips • Pioneer Chip F1 (99-1000-03): Shortened dextran matrix • Large analytes such as cells and virus particles • Pioneer Chip J1 (99-1000-01): Gold surface • Allows design of customized surface chemistry using self-assembled monolayers or other modifications

14. F1 & 2 F1 F3 & 4 F2 F1 - 3 F3 F1 - 4 F4 The Flow Cell Surface is divided into 4 channels, which can be used individually or in a number of combinations.

15. Low reagents consumption Efficient mass transport Low dispersion Highly reproducible injections; CV typically less than 1% Wide range of contact times, 1 s - 12 h Sample recovery and fractionation Microfluidic System

16. Measurement of Binding. • Binding is measured as a change in the refractive index at the surface of the sensor. • This is due to ‘Surface Plasmon Resonance’ (SPR). • The change in refractive index is essentially the same for a given mass concentration change. (allows mass/concentration deductions to be made) • Binding events are measured in real time. (allowing separate on and off rates to be measured.)

17. The Theory behind it. • Binding is measured as a change in the refractive index at the surface of the sensor… How?

18. Total Internal Reflection • At a certain angle of incidence, light entering a prism is totally internally reflected. (TIR). • Although no photons exit the reflecting surface, their electric field extends ~1/4 wavelength beyond the surface.

19. Surface Plasmon Resonance • If a thin gold film is placed on the reflecting surface, the photons can interact with free electrons in the gold surface. • Under the right conditions, this causes the photons to be converted into plasmons and the light is no longer reflected.

20. Surface Plasmon Resonance • This occurs when the incident light vector is equal to the surface plasmon vector….

21. Effect of binding on SPR. • Plasmons create an electric field (evanescant) that extends into the medium surrounding the film. • This is affected by changes in the medium (eg binding of analyte), and results in a change in the velocity of the plasmons. • This change in velocity alters the incident light vector required for SPR and minimum reflection.

22. How does BIACore Measure this? • Fixed wavelength light, in a fan-shaped form, is directed at the sensor surface and binding events are detected as changes in the particular angle where SPR creates extinction of light.

23. The Sensorgram

24. Binding Analysis • How Much? Active Concentration. • How Fast? Kinetics. • How Strong? Affinity. • How Specific? Specificity.

25. Concentration. • Signal proportional to mass. • Same specific response for different proteins.

26. Binding Kinetics • Real-time ‘association’ and ‘dissociation’ rates. • Analysis of bivalent, multimeric and heterogenous analytes. • Analytes from around 340 Da to whole cells.

27. ka kd Binding Kinetics dissociation association

28. Stage One Choose ligand. Choose chip/immobilisation method. Using it...

29. Sensor Chips: CM5 SA NTA HPA Pioneer Chips

30. Stage One Choose ligand. Choose chip/immobilisation method. Using it... • Choose immobilisation levels. • Immobilise ligand.

31. Specificity • Concentration • Affinity • Kinetics • LMW binding Immobilisation Levels. Low High

32. Stage Two Choose analyte. Choose regeneration method. Choose type of analysis required. Using it...

33. BIAControl. Controls the BIACore and records the sensorgram. BIAEvaluation. Kinetic analysis of sensorgrams. BIASimulation. Allows simulation of various binding situations.. BIACore Software.

34. Surface Preparation ‘wizard’ Kinetics analysis ‘wizard’ Manual operation Custom methods (eg. MICRORECOVER) Types of Analysis

35. Immobilising ligand using defined conditions or to a preset target level. Preparing reference surfaces for inline reference subtraction. Testing analyte binding capacity and regenration conditions Surface Preparation Wizard

36. Analysis using captured ligand Determination of kinetic parameters from analyte concentration series Control experiments for mass transfer, linked reactions and kinetic heterogeneity. Kinetic Analysis Wizard

37. Manual setting of : detection mode, flow path flow rates contact times injection volumes… Manual Operation

38. Allows complete control of automated procedures. eg MICRORECOVER method for recovery of bound analyte in small (µl) volumes. Custom Methods

39. BIAControl. Controls the BIACore and records the sensorgram. BIAEvaluation. Kinetic analysis of sensorgrams. BIASimulation. Allows simulation of various binding situations.. BIACore Software.

40. All in a days work! • 9am: Immobilisation of ligand to the sensor chip • 10am: Set up and start analysis wizard • (approx 3 hours per analyte) • 2pm: Use of BIAEvaluation to analyse results • 4pm: Printing of results • Last post: Submission to Nature

41. Further information. Further info and examples can be found under “BIACore” on the “Facilities” page, in the “General Information” section of the AMS website: http://www.ams.rdg.ac.uk/info/facilities.html