LIPID-BINDING ASSAY
Based on a Schiavo protocol (PNAS, 93, 13327-13332)
Buffer A:
20mM Hepes-KOH (pH7.6)
100mM KCl
0.2mM DTT (add DTT just before use).
Liposome matrix:
63%-65% PC (egg lecithin)
20% PS
15%PE
Up to 2% "subject" lipid e.g. PI3P can then be substituted for PC in the mix (->63%PC)
Tracer (L-3-phosphatidyl(N-methyl-3H)choline, 1,2-dipalmitoyl (Amsherham)) at 1µCi per 175µg of lipid (stock: 1.0mCi/ml)
Mix lipid solutions in a clear glass vessel.
Dry under a steady stream of argon (be careful this is not too strong) and resuspend as appropriate (buffer A).
Rehydrate in buffer A with vigorous vortexing-> multilamellar liposomes. 175µg lipid/ml.
Extrude X 10 through 0.1µm pore size polycarbonate filters..
40µl (per experimental point) of glutathione sepaharose bead slurry are washed x3 with buffer A+1mM MgCl2, 1µM ZnCl2 (wash in 15ml tube). The binding capacity of the beads is about 5mg GST per 1ml bed volume (this equivalent to ~7.5mg of a 38.7kD GST-tagged protein.
Assume bed volume=0.5x slurry volume= 20µl per point. therfore we can take 150µg of protein per point maximum ( we will use 100µg).
incubate with beads on the wheel in the coldroom for one hour.
Wash beads x5 with bufferA + 1mM MgCl2 +1µMZnCl2.
Add 100µl of 3H-labelled liposomes to the beads and incubated for 30minutes at room temperature with agitation. Beads alone provide a negative control.
Centrifuge at 1500xg (microfuge) for two minutes and wash three times with buffer A supplemented with 1mM MgCl2.
Solubilise bound lipid by the addition of 0.3ml of 10% SDS to the beads. Spin and carefully take off 200µl. Radioactivity is detected by scintillation counting.