View source for Liposome Binding Assay

Modified from PMID 16829131 

==Materials==
*Br-POPC (Avanti Polar Lipids Cat# 850418P).  Dissolve 10 mg in 1.08 mL of 1:1 CHCl3:MeOH with 0.1% HCl for a 10 mM solution.  Store in glass vial at -80 in 100 uL aliquots.
*Phosphoinositide of interest.  (Avanti Polar Lipids Dioleyl tail groups).  Dissolve 0.5 mg in ~500 uL (depending on Molecular Weight) of 1:1 CHCl3:MeOH with 0.1% HCl for a 1 mM solution.  Store in glass vial at -80 in 100 uL aliquots.
*Nitrogen gas stream
*HBS (25 mM HEPES 7.4, 100 mM NaCl)
*Protein of interest (free of DTT/Glycerol/Glutathione)
*TLA 100 tubes (Beckman Cat# 349622)

==Protocol==
#Mix 100:0 and 97:3 molar ratios of Br-POPC and Br-POPC:PI in a glass vial
#Dry under nitrogen gas then expose to vaccuum for 1-2h to completely dry
#Rehydrate in HBS to a concentration of 25 mM (total lipid)
#Vortex vigorously and freeze/thaw 10-20X in liquid nitrogen/45C.  Should become optically clear.  If it is not clear pass through a 1.0 um extruder until clear.
#Correct pH to 7.2 after 1-2 cycles via spotting on pH paper
#Prepare assay volumes of 50 uL in TLA-100 tubes with ~10 uM protein and varying lipid concentrations (for a first try use 0-1 mM lipid at 10X dilutions)
#Centrifuge at 85 000 RPM for 1h at 25C
#Remove 25 uL of supernatants to fresh tube and aspirate liquid
#Resuspend pellet in 50 uL HBS and sonicated.
#Quantify protein in both pellet and supernatant by BCA assay.  Add SDS to 0.5% to all tubes after BCA but before measuring absorbance to remove scattering of light.
#If protein is limiting, quantify by western blotting.

Modified from PMID 16829131 

[[Category: Lipid Analysis]]
[[Category: Inositol Lipids]]
[[Category: Protein-Lipid Interactions]]