1. EMBO Practical Course: Quantitative FRET, FRAP, and FCS Group 1 (Joana Ferreira, Andreas Diepold), Experiment 1a Single-Pair FRET with TIRF / ALEX setup

2. Calibration of the split camera with fluorescent beads Streptavidin-coating of HF cleaned (!) coverslip Attachment of biotinylated sample Experimental Setup

3. Calibration of the split camera with fluorescent beads Streptavidin-coating of HF cleaned (!) coverslip Attachment of biotinylated sample: a)High FRET control: - Biotinylated double-fluorescently tagged dsDNA Experimental Setup

4. Calibration of the split camera with fluorescent beads Streptavidin-coating of HF cleaned (!) coverslip Attachment of biotinylated sample: a)High FRET control: - Biotinylated double-fluorescently tagged dsDNA b)CAP stabilization of transient DNA interaction: - Biotinylated green-labeled half-site DNA 1 - Red-labeled half-site DNA 1 - cAMP - +/- CAP Experimental Setup

5. Calibration of the split camera with fluorescent beads Streptavidin-coating of HF cleaned (!) coverslip Attachment of biotinylated sample: a)High FRET control: - Biotinylated double-fluorescently tagged dsDNA b)CAP stabilization of transient DNA interaction: - Biotinylated green-labeled half-site DNA 1 - Red-labeled half-site DNA 1 - cAMP - +/- CAP Data analysis Experimental Setup

6. Camera calibration: Fluorescent beads (green channel, bleeding into red channel) Manual assignment of corresponding spots  Transformation matrix (for the complete experiment) Experimental Setup Data analysis: Rotation of pictures Determination of suitable thresholds for the three possible events: D ex /D em, D ex /A em, A ex /A em ; choice of events of interest Automatic analysis of all pictures and matchmaking of corresponding green and red spots  Scatter plot Data acquisition: Focusing on spots Series of pictures (1 sec, 20 fps, alternating green/red excitation)

7. Positive control – High-FRET DNA FRET efficiency: 0.59 - FWHM: 0.1825 Results

8. Transcription Factor Detection

9. Results Negative control: –Half side 1 – no red signal detection –Add half side 2 – transient binding

10. Results CAP –Increase on the red signal, coicindent with green signal –No FRET signal (due to large distance donor - acceptor)

11. Conclusions Biological: CAP stabilizes the interaction of the two halfs of the CAP binding domains Technical: Most useful for immobilized samples (low background noise from mobile molecules).  Single molecule technique  Distinction between subpopulations (temporal and spatial resolution)  Distinction between common movement and direct interaction (method works for non-FRETing samples)  Stoichiometry information  Long measurement periods (up to 30 minutes)

12. Thanks to the tutors! Robert Crawford Alex Kiel Kostas Lymperopoulos Achillefs Kapanidis Dirk-Peter Herten

13. Thanks for your attention! Joana FerreiraAndreas Diepold