FRET is a phenomenon where energy is transferred from one excited molecule to a different molecule through a non-radiative process. Since it is highly dependant on distance, FRET is a powerful tool to assess molecular interactions.
In this application note we will use FRET to follow the binding of a ligand to a protein. By labelling the protein with a FRET donor and the ligand with a compatible FRET acceptor, we can track the interaction using the decrease in the donor signal. The specific concentrations will have to be updated to your protein/ligand pair of interest.
Benefits
- Automatic titration provides accurate titration curves with no hassle
- Measurements and calculations can be automated, saving time and work
- Small volumes can be conveniently handled, saving precious reagents
Workflow
Setting up
Create a new procedure, using the figures below as a starting point. Make any adjustments that may be needed for your experiment. Prepare your reagents.
Run the experiment
Start the experiment procedure and load your reagents. Press ‘Go’.
Get your answers
Find the plots in your Labbot data folder. Process the data and calculate KD
Data
Fig 1: The fluorescence intensity at 570 nm as a function of the total ligand concentration (green dots). As the ligand binds to the protein, fluorescence intensity of the donor is decreased as the energy is transferred to the acceptor and emitted at a longer wavelength.
A fit to the ligand binding equation (red dashes), yielding KD = 250 nM.
Fig 2: Examples of spectra collected at different points throughout the titration. The peak at 570 nm originates from donor and the one at 670 nm is emitted by the acceptor.
Technical Note (PDF)
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Authors
Mattias Törnquist
Helping fellow scientists get the most out of their experiments with Labbot.
Sara Snogerup Linse
Sara is a rockstar in the protein aggregation field. She holds the record for most Labbots in a lab, with her five Labbots operating.