Fluorescence Spectroscopy - the Fluorolog 3.22

FRET experiment

FRET for monitoring the fusion of vesicles induced by membrane peptides :

* Prepare a suspension of SUV at 2 mg/mL with POPC/POPS 3/1 in your Buffer.

* Prepare a suspension of SUV with the same lipids supplemented with ≈ 1% of rhodamine labeled lipids and ≈ 1% of NBD labeled lipids. (visit Avanti Polar Lipids for labeled lipids)

* Make a stock solution by mixing 5% of labeled vesicle with 95% of non-lableled vesicles.

* Adjust the excitation/emission of the spectrometer to 455 nm/465-650 nm, respectively.

* In the cuvette mix some microL of the lipid stock solution with your buffer and record a spectrum.

* Add some peptide solution into the cuvette and record a spectrum to monitor the changes.

Upper image : in blue; FRET for labeled vesicles in presence of the Vectofusin-1 peptide. In red; the same experiment with 3 times more peptides.