User:Tiffany Byerly/Notebook/High Point University - Cannon Lab Research

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Osmolyte Effects on Protein Membranes

  • Objective: To determine the K of the binding of large unimellar vesicles to peptides in solution

Preparation Steps

Buffer Preparation

  • 0.1M sodium phosphate buffer at pH 7
  • Need:
  1. 30.5mL of 0.2M Na2HPO4*2H2O
  2. 19.5mL of 0.2M Na2H2PO4*2H2O
  3. Dilue with DI H2O to 100mL
  4. Actual pH 6.94

Lipid Film Preparation

  1. Prepare 4mL of 3:1 CHCl3:Methyl Alcohol
    • 3mL CHCl3
    • 1mL Methyl Alcohol
  2. Add 2.5mL solution to 1 lipid vial (m = 25mg; mw = 760.10g/mol)
  3. Place 1mL lipid solution in 4mL glass vial, mix
  4. Dry on rotoevaporator
  5. Place aliquot in desicator and store overnight
  6. Store lipid film at room temperature

Lipid Vesicle Preparation

  1. Add 3.5mL of buffer (see above) to vial
    • Vesicle conc. = 9.428mM
  2. Agitate for at least 2 hours
  3. Using mini-extruder, prepare large unilamellar vesicles in solution
  4. Store vesicles at room temperature

Dilute Lipid Vesicle Solution Preparation

  1. Using lipd vesicle solution and buffer, dilute vesicle solution to concentrations of 10µM, 30µM, and 50µM (1mL total solution)

Peptide Solution Preparation

  1. Dissolve 9.6e-5g peptide (mw = 642.79g/mol)in 1mL buffer
  2. Using UV spectrophotomer and Beer's Law, calculate the concentration of the peptide solution
    • ε = 5,579 M−1 cm−1
    • Peptide conc. = 750µM

Dilute Peptide Solution Preparation

  1. Using peptide solution and buffer, dilute peptide solution to concentrations of 2µM, 10µM, and 30µM (1mL total solution)