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

  1. 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
  2. Controls
    • Obtain a baseline with the following:
      • sodium phosphate buffer
      • peptide solution of all concentrations
      • lipid solution of all concentrations
  3. 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

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