Protonation of phosphoinositides using acid

Protocol from Olga Perisic, R. Williamsí lab, MRC

Resuspension of PI(3,4)P2 powder in CHCl3 is not sufficient for efficient incorporation into liposomes. It is essential that the lipid is previously sufficiently protonated before use in assays. All resuspensions to 2.5 mM lipid conc.

  1. Resuspend PI(3,4)P2 powder in CHCl3.Dry under argon, and dessicate for 1 hour.
  2. Resuspend with ChCl3:MeOH: 1N HCl in molar ratio of 2:1:0.01. Let stand for 15 minutes. Dry under Argon, and dessicate for 1 hour.
  3. Resuspend with ChCl3: MeOH (3:1). Dry under Argon.
  4. Resuspend with ChCl3. Dry under Argon.
  5. Resuspend with ChCl3. Store at ñ70 deg. C.