EMBO Practical Course: Quantitative FRET, FRAP, and FCSGroup 1 (Joana Ferreira, Andreas Diepold), Experiment 1a

Single-Pair FRETwith TIRF / ALEX setup

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

Experimental Setup

• 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

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Experimental Setup

• 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

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Experimental Setup

• 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

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Experimental Setup

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:

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

Data acquisition:• Focusing on spots• Series of pictures

(1 sec, 20 fps, alternating green/red excitation)

• Positive control – High-FRET DNA

FRET efficiency: 0.59 - FWHM: 0.1825

Results

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Results

• Transcription Factor Detection

Results• Negative control:

– Half side 1 – no red signal detection

– Add half side 2 – transient binding

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Results• CAP

– Increase on the red signal, coicindent with green signal

– No FRET signal (due to large distance donor - acceptor)

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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)

Thanks to the tutors!

Robert CrawfordAlex KielKostas LymperopoulosAchillefs KapanidisDirk-Peter Herten

Thanks for your attention!

Joana Ferreira Andreas Diepold

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