Difference between revisions of "Liposome Binding Assay"

Latest revision as of 13:01, 2 November 2010

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

1. Mix 100:0 and 97:3 molar ratios of Br-POPC and Br-POPC:PI in a glass vial
2. Dry under nitrogen gas then expose to vaccuum for 1-2h to completely dry
3. Rehydrate in HBS to a concentration of 25 mM (total lipid)
4. 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.
5. Correct pH to 7.2 after 1-2 cycles via spotting on pH paper
6. 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)
7. Centrifuge at 85 000 RPM for 1h at 25C
8. Remove 25 uL of supernatants to fresh tube and aspirate liquid
9. Resuspend pellet in 50 uL HBS and sonicated.
10. 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.
11. If protein is limiting, quantify by western blotting.

Modified from PMID 16829131