Difference between revisions of "Surface Plasmon Resonance - Protein Lipid Interactions"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (Created page with '==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 ...') |
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#Inject 20 uL 0.1M NaOH between samples | #Inject 20 uL 0.1M NaOH between samples | ||
#For Kd determination, inject buffer 2-3x first to get a blank reading | #For Kd determination, inject buffer 2-3x first to get a blank reading | ||
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+ | ==Reference== | ||
+ | <pubmed>16829131</pubmed> |
Revision as of 00:39, 2 May 2009
Contents
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
- Combine DOPC + lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen (700uL PC or 679uL PC + 21 uL PI)
- Resuspend to 10 mM total lipid with HBS-N (700 uL). Vortex thoroughly and sonicate in water bath
- Correct pH to 7.4 using pH paper and 1uL aliquots of 1M NaOH
- Freeze thaw in a water bath sonicator at 40C and liquid nitrogen 8 times.
- Pass 10X through a polycarbonate filter using an Avanti MiniExtruder
- Dilute to 1.5 mM total lipid with HBS-N (final volume of 105 uL)
Preparation of Surface
- Wash all four surfaces at 10 uL/min and 25C with HBS-N
- Inject 20 uL of 1% beta-octylglucoside
- Inject 20 uL of 0.5% SDS
- Inject 10 uL of 1% beta-octylglucoside
- Inject 10 uL of 30% ethanol
- 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)
- Wash with 20 uL of 0.1M NaOH
Analysis of Sample
- Inject protein samples adjusting contact time as necessary to reach saturation (typically 400s per injection)
- Inject 20 uL 0.1M NaOH between samples
- For Kd determination, inject buffer 2-3x first to get a blank reading
Reference
<pubmed>16829131</pubmed>