Parasite Synchronization and Protein Extraction

Parasite cultures were synchronized to the ring stage by the 5% sorbitol method [28 (link)] and incubated for approximately 24 hours until parasites developed to the trophozoite stage. iRBCs were incubated with 0.2 mg/mL proteinase K in incomplete medium (ICM) (Ht 12.5%) at 37°C for 15 min using a tube rotator (MACSmix; Miltenyi Biotec, Germany), washed once with ICM, and then incubated with 4 mM Pefabloc solution (Roche, Switzerland) at 37°C for 2 hours to inactivate proteinase K. Proteins were extracted from parasite-iRBC essentially as described above except that the parasite pellets were treated with 2% SDS after the freezing and thawing process. Additionally, PBS containing cOmplete, 1 mM EDTA and 4 mM Pefabloc was used instead of PBS/PI-EDTA. Finally, protein extracts corresponding to 3.8 × 10⁷ parasite/lane were separated using 5–20% gradient SDS-polyacrylamide gels.