1        MATERIALS

1.1       Parasite cultures (~250 mL of 8-12% parasitemia cultures)

1.2       15 mL and 50 mL sterile tubes

1.3       Phosphate-buffered saline (PBS) (Gibco)

1.4       1.5% Saponin stock solution (Acros Organics)

Note: important… To work on ubiquitinated proteins, 20 mM N-ethyl maleimide and 10 mM EDTA should be added to the PBS buffer.

2        PROCEDURE

2.1       Resuspend parasite culture and transfer to 50 mL sterile tubes.

2.2       Centrifuge 800 g for 5 min @ 4°C.

2.3       Remove the supernatant and bring volume to 40 mL using PBS. Resuspend the cells.

2.4       Centrifuge 800 g for 5 min @ 4°C.

2.5       Remove the supernatant and add 5 pellet volumes of 0.15% Saponin, mix by pipetting until the color changes to a dark red indicating red blood cell lysis.

2.6       Leave on ice 10-15 min after homogenization.

2.7       Centrifuge 3200 g for 10 min @ 4°C.

2.8       Remove the supernatant and add 2 pellet volumes of 0.15% Saponin, then bring volume to 40 mL total using PBS. Mix slowly by pipetting and leave on ice 5 min.

2.9       Centrifuge 3200 g for 10 min @ 4°C.

2.10    Combine pellets into one 15 mL sterile tube and bring volume to 15 mL using PBS.

2.11    Centrifuge 3200 g for 10 min @ 4°C.

2.12    Remove the supernatant and aliquot pellet in microcentrifuge tubes.

2.13    Fill centrifuge tubes with PBS and centrifuge 3200 g for 5 min @ 4°C.

2.14    Remove supernatant and freeze parasite pellets on liquid nitrogen while in microcentrifuge tubes. The final pellet of pure parasites should be black and 250 mL - 1 mLin size depending on amount and parasitemia of starting culture.

2.15    Store parasite pellets @ -80°C.