1. detect and identify Bioanalytic Biomolecular Interactions, Manchester, June 2009 Biomolecular Applications with Mithras and TriStar multimode plate readers

2. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Basic information Mithras and TriStar FRET: CFP/YFP Fluorescence Polarisation: nuclear receptor HTRF ® : multiplexing BRET: receptor dimerisation PathHunter™: GPCR

3. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Basics Luminescence „… is light that usually occurs at low temperatures, and is thus a form of cold body radiation. It can be caused by chemical reactions, electrical energy, …“ (Wikipedia)

4. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Basics types of luminescence: Bioluminescence Chemo/Chemiluminescence Mechanoluminescence Thermoluminescence Radioluminescence Photoluminescence Phosphorescence Fluorescence

5. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Basics Fluorescence Prompt Fluorescence Fluorescence Polarisation Time-Resolved Fluorescence Fluorescence Lifetime

6. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Basics Fluorescence „… is a luminescence that is mostly found as an optical phenomenon in cold bodies, in which the molecular absorption of a photon triggers the emission of a photon with a longer (less energetic) wavelength. The energy difference between the absorbed and emitted photons ends up as molecular rotations, vibrations or heat.“ (Wikipedia)

7. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Basics Fluorescence: Jablonski diagram

8. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Basics Fluorescence: Stokes shift λ em stays

9. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Selecting Filters

10. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Selecting filters

11. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Selecting filters

12. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Selecting filters Overlap 

13. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Excitation and emission filter pairs must not to overlap with their transmission spectra! Selecting filters

14. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Berthold Filter Data Base Selecting filters

15. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Web tools Invitrogen Spectra Viewer http://www.invitrogen.com/site/us/en/home/support/ Research-Tools/Fluorescence-SpectraViewer.html Omega Curv-O-matic https://www.omegafilters.com/curvo2/index.php Fluorophores.org http://www.analytchem.tugraz.at/fluorophores/Browse Substance.php Selecting filters

16. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Mithras & Tristar

17. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Mithras & TriStar Luminescence BRET/BRET² AlphaScreen™ Absorbance Prompt Fluoresc. FRET Fluor. Polarisation Time-Resolved Fl. HTRF ® Luminescence BRET/BRET² Absorbance Prompt Fluoresc. FRET

18. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Mithras & TriStar DOPS D edicated O ptical P ath S ystem

19. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Mithras & TriStar Patented cross-talk reduction device Aperture wheel 10 -6 in black plates

20. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Mithras & TriStar Photon Counting PMT operation Stringent PMT detector selection

21. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Mithras & TriStar Reagent Injectors: JET technology Teflon bellows No friction Cell-friendly

22. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Mithras & TriStar Reagent Injectors: JET technology Fast and efficient mixing

23. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Mithras & TriStar Reagent Injectors: JET technology Highest precision and accuracy

24. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FRET CFP/YFP

25. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FRET CFP/YFP Spectral overlap of donor em and acceptor ex spectra Close proximity 1 – 10 nm Förster distance

26. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FRET CFP/YFP 2 readings Ratio calculation

27. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FRET CFP/YFP JBC paper, June 2006, Nikolaev et al. Partial agonism at the α 2A adrenergic receptor Heterotrimers G i type

28. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FRET CFP/YFP cAMP monitoring with Epac2 JBC, June 2006, Nikolaev et al. and JBC, June 2004, Nikolaev et al.

29. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FRET CFP/YFP cAMP monitoring with Epac2 JBC, June 2006, Nikolaev et al. and JBC, June 2004, Nikolaev et al.

30. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FRET CFP/YFP cAMP monitoring with Epac2 HEK293 cells incubated for 10 min with agonist Ethanolic cAMP extraction, drying, desolving in TRIS buffer Black 96-well plate 50 µL cAMP solution 50 µL Epac-camps (final 0.6 ng/µL) 1 st read ex 430 nm, em 480 nm 2 nd read ex 430 nm, em 535 nm JBC, June 2006, Nikolaev et al.

31. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FRET CFP/YFP cAMP monitoring with Epac2 cAMP dose-response JBC, June 2006, Nikolaev et al.

32. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FRET CFP/YFP cAMP monitoring with Epac2 NE (norepinephrine): full agonist JBC, June 2006, Nikolaev et al.

33. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Fluor. Polarisation

34. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Excited state has a lifetime (ns range) During this time the unbound fluorophore rotates and the polarisation of the emitted light differs from the excitation plane Fluor. Polarisation

35. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Fluor. Polarisation 1 st reading: parallel orientation 2 nd reading perpendicular orientation

36. Bioanalytic Biomolecular Interactions, Manchester, June 2009 benefits homogeneous only one labelled partner necessary direct probe for binding detection ratio reading eliminating assay variation influences kinetics possible Cheap limitations low sensitivity needs fairly high amount of binding partner (e.g. receptors) readout strongly dependent on filling height and surface Fluor. Polarisation

37. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FP Receptor-Ligand Binding ER α 50 nM FITC tagged short peptide bearing the nuclear receptor box 2-binding motif of the co-activator protein SRC-1 Titration of receptor

38. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FP Receptor-Ligand Binding Optimum peptide concentration 50 nM

39. Bioanalytic Biomolecular Interactions, Manchester, June 2009 FP Receptor-Ligand Binding Scatchard analyis: K d 7.5 nM

40. Bioanalytic Biomolecular Interactions, Manchester, June 2009 HTRF ®

41. Bioanalytic Biomolecular Interactions, Manchester, June 2009 HTRF ®

42. Bioanalytic Biomolecular Interactions, Manchester, June 2009 HTRF ®

43. Bioanalytic Biomolecular Interactions, Manchester, June 2009 HTRF ® multiplexing IP1 and cAMP

44. Bioanalytic Biomolecular Interactions, Manchester, June 2009 HTRF ® multiplexing IP1 and cAMP

45. Bioanalytic Biomolecular Interactions, Manchester, June 2009 HTRF ® multiplexing IP1 and cAMP

46. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Bio- / Chemi-Luminescence Most efficient light collection Most efficient avoidance of crosstalk

47. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Chemiluminescence Enzyme Fragment Complementation ß-Galactosidase fragment fused to receptor (“Prolink tag”) Enzyme fused to ß-Arrestin Kaiser et al., Greiner

48. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Chemiluminescence Kaiser et al., Greiner

49. Bioanalytic Biomolecular Interactions, Manchester, June 2009 BRET Substrate: Coelenterazine 1 – 5 µM

50. Bioanalytic Biomolecular Interactions, Manchester, June 2009 BRET: receptor dimerisation HEK293 transfected with cDNA encoding CCR4 and CXCR5 receptors fused to hRluc and YFP Issad, Jockers, Marullo, Inst. Cochin

51. Bioanalytic Biomolecular Interactions, Manchester, June 2009 BRET: receptor dynamics Issad, Jockers, Marullo, Inst. Cochin HEK cells were co- transfected with the cDNA coding for IR- Rluc and YFP- PTP1B-D181A tyrosine phosphatase

52. Bioanalytic Biomolecular Interactions, Manchester, June 2009 BRET: receptor conform. changes Issad, Jockers, Marullo, Inst. Cochin Co-expression of MT1-Rluc and MT1-YFP or MT2-Rluc and MT2-YFP

53. Bioanalytic Biomolecular Interactions, Manchester, June 2009 Thank you for your attention Bernd Hutter Bernd.Hutter@Berthold.com www.Berthold.com/bio