Phosphoinositides extraction

Several procedures were described to extract the polyphosphoinositides since they are known to bind strongly to proteins during their denaturation. To improve the recovery, the use of an acidic solution is necessary. We used the procedure of Honeyman (Biochem J 1983, 212, 489) which is a modification of the Lloyd’s method (Br J Haematol 1972, 23, 571-585).

This is the one-step method of Bligh and Dyer modified by the inclusion of HCl to improve recovery of acidic phospholipids :

1 ml of cell suspension is mixed with 3.75 ml of chloroform/methanol/12N HCl (2/4/0.1, v/v). After thorough mixing, 1.25 ml of chloroform is added with vortexing 30 sec followed by 1.25 ml of water with similar mixing. After centrifugation 10 min at low speed, the lower chloroform layer is removed and transferred to a glass tube for evaporation.

Method used to extract platelet phosphoinositides

Stop incubation of the cell suspension by adding 1 ml of chloroform/methanol (1/1) to 1.5 ml of aqueous medium and vortex some seconds. Transfer the mixture with a Pasteur pipette into a 15 ml Falcon polypropylene tube.
Add to the mixture 4 ml of chloroform/methanol (1/1), then 0.4 ml 10N HCl and then 0.5 ml water. Vortex during 5 min to extract lipids and centrifuge the plastic tube at low speed 10 min at 4°C.
Transfer the lower phase into a second Falcon tube by sampling through the proteinaceous disk with a Pasteur pipette. Add to this solution 2.5 ml methanol, 2.1 ml water and 0.4 ml 10N HCl. Vortex some seconds and centrifuge at low speed in the cold.
Transfer the lower phase as previously in a glass tube, evaporate with the help of nitrogen and dissolve the residue with a small volume of chloroform/methanol (1/1).