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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 ...'
==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
*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