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EMBO Practical Course: Quantitative FRET, FRAP, and FCS

EMBO Practical Course: Quantitative FRET, FRAP, and FCS Group 1 (Joana Ferreira, Andreas Diepold), Experiment 1a. Single-Pair FRET with TIRF / ALEX setup. Experimental Setup. Calibration of the split camera with fluorescent beads Streptavidin-coating of HF cleaned (!) coverslip

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EMBO Practical Course: Quantitative FRET, FRAP, and FCS

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

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

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

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

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

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

  8. Results • 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! Andreas Diepold Joana Ferreira

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