20
edits
Changes
m
Additions from protocol handwritten by Dave.
*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
==Preparation of Liposomes==
#Combine DOPC + C16 lipid (or DOPC alone) at a ratio of 97:3 and dry under nitrogen (700uL 150uL PC or 679uL 145.5uL PC + 21 4.5 uL PI)#Resuspend to 10 mM total lipid with HBS-N (700 100 uL). Vortex thoroughly and sonicate in water bath#Correct pH to 7.4 using pH paper and 1uL 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#Dilute to 1at Room Temperature.5 mM total lipid *Rinse syringe with HBS-N (final volume 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 105 uL)milliQ water through the filger ~5 times. *Discard the water and inject the sample through the filter 10 times.
==Preparation of Surface==
#Inject 10 uL of 1% beta-octylglucoside
#Inject 10 uL of 30% ethanol
#Inject 55 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
