Difference between revisions of "Surface Plasmon Resonance - Protein Lipid Interactions"
From Bridges Lab Protocols
m (Additions from protocol handwritten by Dave.) |
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==Preparation of Liposomes== | ==Preparation of Liposomes== | ||
| − | #Combine DOPC + C16 lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen ( | + | #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) |
#Resuspend to 10 mM total lipid with HBS-N (100 uL). Vortex thoroughly and sonicate in water bath | #Resuspend to 10 mM total lipid with HBS-N (100 uL). Vortex thoroughly and sonicate in water bath | ||
#Correct pH to 7.4 using pH paper and 0.6uL aliquots of 1M NaOH | #Correct pH to 7.4 using pH paper and 0.6uL aliquots of 1M NaOH | ||
| Line 24: | Line 24: | ||
*Tightly attach syringe and place on blcok. | *Tightly attach syringe and place on blcok. | ||
*Pass 250uL of milliQ water through the filger ~5 times. | *Pass 250uL of milliQ water through the filger ~5 times. | ||
| − | *Discard the water and inject the sample through the filter 10 times. | + | *Discard the water and inject the sample through the filter 10 times. |
| − | + | ||
==Preparation of Surface== | ==Preparation of Surface== | ||
Revision as of 14:06, 10 June 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)(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
- 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)
- Resuspend to 10 mM total lipid with HBS-N (100 uL). Vortex thoroughly and sonicate in water bath
- Correct pH to 7.4 using pH paper and 0.6uL 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 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
- 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 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)
- 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>
