Surface Plasmon Resonance - Protein Lipid Interactions

From Bridges Lab Protocols
Revision as of 14:07, 10 June 2009 by Jpecherer (Talk | contribs) (→‎Materials)

Jump to: navigation, search

Materials

  • L1 Sensor Chip (Biacore)
  • 10x Dioleylphosphatidylcholine (Sigma) dissolved in 1:1 chloroform:methanol with 0.1% HCl
  • 10x 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)(6.25mL Hepes (1M), 9.38mL NaCl (4M)) Total volume 250mL.
  • 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 + C16 lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen (15uL PC or 14.5uL PC + 0.5 uL PI)
  2. Resuspend to 10 mM total lipid with HBS-N (100 uL). Vortex thoroughly and sonicate in water bath
  3. Correct pH to 7.4 using pH paper and 0.6uL 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 at Room Temperature.
  • Rinse syringe with milliQ water.
  • Wet filter paper and put the filter on the block.
  • Place PC filter on block.
  • Tightly attach syringe and place on blcok.
  • Pass 250uL of milliQ water through the filger ~5 times.
  • Discard the water and inject the sample through the filter 10 times.

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 70 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>

Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php?title=Surface_Plasmon_Resonance_-_Protein_Lipid_Interactions&oldid=183"