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:
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
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.
- Add 0.001g peptide to 1mL sodium phosophate buffer. The pH should be ≈ 7.0.
- Use a UV spectrophotometer to measure the absorbance of the peptide solution at 280nm.
- 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
- Once the concentration of the stock peptide solution is known, make 1mL dilute peptide solution in sodium phosphate buffer (2μM, 10μM, and 30μM peptide).