Sucrose-loaded Vesicle Assay

A. Generate 100% PA, 100% PS, and 100% PC vesicles

B. Assess binding of PP1cg to PA by varying mol% PA in PC or PS vesicles

A. Generation of SLUV (Sucrose-loaded Large Unilaminar Vesicles):

1. Dry lipids in glass sonication vials.

a. 72.2 µl PA, 78.6 µl PC, 81.0 µl PS (equivalent of 1 mM phospholipid in 1 ml volume)

b. Dry under nitrogen (rotate tube continuously to form a thin even film)

2. Add 1 ml of 5mM HEPES, 20mM KCl, 180mM sucrose, pH 7.4, let stand for 30 minutes. This generates a 1 mM phospholipid suspension.

3. Vortex for 1 x 1 minute

4. Freeze/thaw 5 times by freezing in dry-ice/MeOH bath and thawing at 37ūC

5. Pass through Avanti mini-Extruder (with 0.1 micron pore filter) 21 times.

6. Centrifuge in table-top ultracentrifuge at 100,000 x g for 1 hr.

7. Discard supernatant and resuspend pellet in 1 ml of 25 mM HEPES,

100mM KCl, pH 7.4. (Final concentration = 1 mM)

8. Store at 4ūC for up to 3 days in siliconized screw-cap 1.5 ml eppindorf tubes.

B. 6XHis-PP1cg binding to PA, PS and PC by SLUV Binding Assay:

1. Combine in a Beckman ultracentrifuge rated 1.5 ml eppindorf tubes: 25 mM HEPES, 100 mM KCl, pH 7.4, 6XHis-PP1cg at various concentrations (100, 250, 500, and 750 ng), and 500 µM PA, PS or PC SLUV, in a total volume of 50 µl.

2. Incubate at 30ūC for 15 minutes

3. Spin in table-top ultracentrifuge (TLA45 rotor at 45,000 rpm) for 1 hour at 25 ūC

(100000 x g).

4. Collect supernatant and add 4X Laemmli sample buffer. Boil and freeze or run directly for Western blot with anti-6XHis antibody.

5. Resuspend pellet with 1X Laemmli sample buffer. Boil and freeze or run directly for Western blot analysis with anti-6XHis antibody.