BCH4160/2011:Notebook/Jigeshs Lab Notebook 2011/2011/11/01

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Binding Fluorescence Continued The goal of this experiment was to measure the binding fluorescence of a large unilamellar lipid vesicle/peptide solution in order to measure the K of the reaction. Materials Refer back to part one of this experiment. Procedure Preparation of the Lipid Vesicles Add 3mL of the buffer into the pre-made lipid vesicles Vortex for 1 hour Mass of lipids = 0.025j 760.10 g/mol Concentration = 9.39 mM Extrusion of the lipid solution (method used to get unilamellar vesicles) Dilute stock lipid to 5μL, 25μL, and 100μL Controls Obtain a baseline with the following: sodium phosphate buffer peptide solution of all concentrations lipid solution of all concentrations Testing 1:1 dilution of each concentration at 25 °C Total of 9 combinations of lipid-peptide solutions Solution of 5μL lipids with 2μL peptide, 5μL lipids with 10μL peptide solution, and 5μL lipids with 30μL peptide solution Solution of 25μL lipids with 2μL peptide, 25μL lipids with 10μL peptide solution, and 25μL lipids with 30μL peptide solution Solution of 100μL lipids with 2μL peptide, 100μL lipids with 10μL peptide solution, and 100μL lipids with 30μL peptide solution Data & Results Contact Who has experience with this protocol? or instead, discuss this protocol.