embo practical course: quantitative fret, frap, and fcs group 1 (joana ferreira, andreas diepold),...
TRANSCRIPT
EMBO Practical Course: Quantitative FRET, FRAP, and FCSGroup 1 (Joana Ferreira, Andreas Diepold), Experiment 1a
Single-Pair FRETwith TIRF / ALEX setup
Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“
benötigt.
• 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
Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“
benötigt.
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
Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“
benötigt.
Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“
benötigt.
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
Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“
benötigt.
Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“
benötigt.
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
Zur Anzeige wird der QuickTime™ Dekompressor „Sorenson Video“
benötigt.
Results
• Transcription Factor Detection
Results• Negative control:
– Half side 1 – no red signal detection
– Add half side 2 – transient binding
Zur Anzeige wird der QuickTime™ Dekompressor „Sorenson Video“
benötigt.
Results• CAP
– Increase on the red signal, coicindent with green signal
– No FRET signal (due to large distance donor - acceptor)
Zur Anzeige wird der QuickTime™ Dekompressor „Sorenson Video“
benötigt.
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
Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“
benötigt.
Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“
benötigt.
Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“
benötigt.