1        MATERIALS

1.1       Lysis buffer

·      20 mM Tris HCl 7.5

·      20 mM NEM

·      20 mM EDTA

·      Complete Mini EDTA-free protease inhibitor mixture (Roche Applied Sciences; #04693159001)

o      7X stoch solutions are prepared by dissolving one tablet in 1.5 mL redistilled water or in 100 mM phosphate buffer;

o      1.5 mL of stock solution is added to 8.5 mL of the lysis buffer without protease inhibitor)

1.2       Biorad dye reagent

1.3       SDS PAGE Gel

2        PROCEDURE

2.1       Resuspend parasite pellet in ~2 volumes of ice-cold lysis buffer

2.2       Homogenize by brief sonication @ 4°C (6 ´ 10-20 s, 20% sonication max, until the color turns grey/black)

2.3       Centrifuge 15 min @ 4°C and 20,000 g (max speed)

2.4       Supernatant is soluble fraction (cytoplasmic proteins), pellet is insoluble fraction (membrane proteins)