1. detect and identify Bioanalytic Bioluminescence Resonance Energy Transfer

2. Bioanalytic What is BRET ? Energy (light) of Renilla reaction is transferred to fluorescent protein Read-out at emission wavelength of fluorescent protein Occurs upon close proximity Due to interaction of attached biomolecules

3. Bioanalytic 7 transmembrane receptor = G protein coupled receptor (GPCR) Renilla luciferase Based on an example of GPCR: This kind of assay is performed with whole cells How does BRET work ?

4. Bioanalytic 7 transmembrane receptor = G protein coupled receptor (GPCR) R. luciferase Upon addition of the Renilla luciferase substrate coelenterazine blue light with a peak at 480 nm is emitted Coelenterazine 1 to 5 µM

5. Bioanalytic ligand / agonist Attachment of ligand causes activation of the receptor incl. dissociation of the G-protein subunits and phosphorylation of the receptor R. luciferase Coelenterazine 1 to 5 µM

6. Bioanalytic R. luciferase Coelenterazine 1 to 5 µM ß-arrestin/eYFP ß-arrestin/eYFP fusion protein attaches to activated GPCR; this results in proximity of the Renilla luciferase and the eYFP moiety, close enough for the emitted light of the luciferase reaction to cause an energy transfer upon which eYFP emits light at ist specific emission wavelength

7. Bioanalytic Spectra Proc. Natl. Acad. Sci. USA Vol. 96, pp. 151–156, January 1999 Cell Biology

8. Bioanalytic Applications GPCR functional assay incl. orphan receptors Receptor oligomerisation Membrane reorganisation Protein-protein interactions e.g. interaction of circadian clock proteins Caspase etc.

9. Bioanalytic Dimerisation of receptors within a membrane

10. Bioanalytic Interaction of KaiB proteins Proc. Natl. Acad. Sci. USA Vol. 96, pp. 151–156, January 1999 Cell Biology

11. Bioanalytic Caspase PerkinElmer Life Sciences Application Note BRT001

12. Bioanalytic Why use BRET ? No photo-bleaching or photo-isomerization of the donor protein or auto-fluorescence from cells or microplates caused by incident excitation light

13. Bioanalytic Non-destructive homogeneous assay: coelenterazine is membrane permeable and no filtration nor wash steps are required

14. Bioanalytic Functional assay for GPCR research and screening as only "real" agonists and antagonists affect changes in BRET signal

15. Bioanalytic Universal assay for GPCRs as almost all known receptors bind ß-arrestin no matter if G s, G i or G q Gsstimulating cAMP production Giinhibiting cAMP production Gqstimulating Inositolphosphatase-3 pathway

16. Bioanalytic Signal stability: Although total signal decreases over time, BRET ratios are stable for a period of at least one hour in cells

17. Bioanalytic Ratiometric signal: minimal interference from cell type and number, microplate variability, etc.

18. Bioanalytic BRET occurs rapidly upon addition of coelenterazine, resulting in the potential for kinetic analysis

19. Bioanalytic Assay versatility: BRET can be used in different applications, e.g. protein-protein interaction, fusion protein configuration, GPCRs, etc.

20. Bioanalytic No labeling: „labels“ are proteins which are co-expressed in the cells

21. Bioanalytic Non-radioactive: HTS technologies like Flashplate or SPA are based on radiolabeled tracers

22. Bioanalytic BRET pairs DonorAcceptor R. lucGFP R. luceYFP R. luc with DeepBlueCGFP (Packard) R. luc with AequorinGFP R. luc with ?dsRed

23. Bioanalytic BRET performance

24. Bioanalytic BRET dose response

25. Bioanalytic