BCH4160/2011:Notebook/Laura Lee/2011/10/25

Binding Fluorescence Studies

The purpose of this experiment was to measure the fluorescence of lipid-peptide binding at different concentrations of lipids and peptide. This experiment is a continuation of the studies done by other High Point University students. The prior protocols are listed below:

• [Byerly's protocol]
• [Tindall's protocol]

Materials

Materials used in this experiment:

• Lipid films made using this protocol
• Peptide (MW = 642.79g)
• Sodium phosphate buffer (made using prior buffer protocol)
• Fluorescence spectrophotometer

Procedure

Preliminary Preparations of Peptide Solutions

The purpose of protocol is to prepare peptide solutions of concentrations 2μM, 10μM, and 30μM for future binding fluorescence lab.

1. Add 0.001g peptide to 1mL sodium phosophate buffer. The pH should be ≈ 7.0.
2. Use a UV spectrophotometer to measure the absorbance of the peptide solution at 280nm.
3. Calculate the concentration of the peptide solution using the measured absorbance and Beer's Law.
   • Beer's Law: A=εlc → c = (A÷(εl))
   • ε=5500 M-1 cm-1
   • Wavelength = 280 nm
   • l = 0.2cm
4. Once the concentration of the stock peptide solution is known, make 1mL dilute peptide solution in sodium phosphate buffer (2μM, 10μM, and 30μM peptide).

Data/Results

Refer to part 2 of the Fluorescence Binding experiment.

Contact

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