BCH4160/2011:Notebook/Laura Lee/2011/11/01

Binding Fluorescence Study Continued

This protocol is a continuation of binding fluorescence study part one.

Materials

Refer back to binding fluorescence study part one.

Procedure

Refer back to this for prior part of protocol.

Preparation of Unilamellar Lipid Vescles

The purpose of this part of the experiment was to rehydrate our previously made lipid films and use them to make unilamellar vesicles.

1. Add 3mL of sodium phosphate buffer to previously dried lipid films to rehydrate.
   • Note: characteristics of lipids are:
     • Mass: 0.025g
     • Molecular weight: 760.10 g/mol
     • Concentration: 9.39mM
2. Vortex the vial for one hour.
3. Use extrusion methods on the stock lipid solution to achieve unilameller vesicles.
4. Dilute samples to the following concentrations: 5μM, 10μM and 100μM.

Controls of Experiment

1. Use sodium phosphate buffer as a blank with a fluorescence spectrophotometer.
2. Measure fluorescence graph baselines of all final concentrations of lipids and peptides by themselves. (Essentially take the original peptide concentrations made previously and dilute them 1:1 with buffer, as they will be 1:1 when the lipids and peptides are combined).
   • Final peptide concentrations: 1μM, 5μM, 30μM
   • Final lipid concentrations: 2.5μM, 12.5μM, 50μM

Testing

1. Measure fluorescence graphs of 1:1 dilutions of each concentration at 25°C.
   • Sample combinations of previously diluted peptide and lipid solution:
     • 5μM lipid + 2μM peptide
     • 5μM lipid + 25μM peptide
     • 5μM lipid + 100μM peptide
     • 25μM lipid + 2μM peptide
     • 25μM lipid + 25μM peptide
     • 25μM lipid + 100μM peptide
     • 100μM lipid + 2μM peptide
     • 100μM lipid + 25μM peptide
     • 100μM lipid + 100μM peptide

Data/Results

Raw Data--> Experiment: File:Fluorescence.xls. Baseline (of just peptide and lipid concentrations): File:Baseline of basic peptide and lipid concentrations.xls

Contact

This experiment was done with High Point University students Jigesh Patel and Brittany Webster.