Surface Plasmon Resonance - Protein Lipid Interactions

Materials

• L1 Sensor Chip (Biacore)
• Dioleylphosphatidylcholine (Sigma) dissolved in 1:1 chloroform:methanol with 0.1% HCl
• PIPx of interest (Avanti or Echelon) dissolved in 1:1 chloroform:methanol with 0.1% HCl
• HBS-N (25mM HEPES, pH 7.4, 150 mM NaCl)
• 1M NaOH
• pH strips
• Water bath sonicator set to 40C
• Avanti MiniExtruder
• 1% beta octylglucoside
• 0.5% SDS
• 30% ethanol
• 100mM NaOH

Preparation of Liposomes

1. Combine DOPC + lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen (700uL PC or 679uL PC + 21 uL PI)
2. Resuspend to 10 mM total lipid with HBS-N (700 uL). Vortex thoroughly and sonicate in water bath
3. Correct pH to 7.4 using pH paper and 1uL aliquots of 1M NaOH
4. Freeze thaw in a water bath sonicator at 40C and liquid nitrogen 8 times.
5. Pass 10X through a polycarbonate filter using an Avanti MiniExtruder
6. Dilute to 1.5 mM total lipid with HBS-N (final volume of 105 uL)

Preparation of Surface

1. Wash all four surfaces at 10 uL/min and 25C with HBS-N
2. Inject 20 uL of 1% beta-octylglucoside
3. Inject 20 uL of 0.5% SDS
4. Inject 10 uL of 1% beta-octylglucoside
5. Inject 10 uL of 30% ethanol
6. Inject 55 uL of extruded lipid suspension at 10uL/min (should see an increase of 5000-10000RU) to desired well (start with lane 4 and move to lane 1-PC only, lipids can migrate so load in a way to minimize contamination affecting results)
7. Wash with 20 uL of 0.1M NaOH

Analysis of Sample

1. Inject protein samples adjusting contact time as necessary to reach saturation (typically 400s per injection)
2. Inject 20 uL 0.1M NaOH between samples
3. For Kd determination, inject buffer 2-3x first to get a blank reading

Reference

<pubmed>16829131</pubmed>