Ookinete Microneme Protein Secretion

Microneme secreted proteins were examined in in vitro ookinete culture supernatants as previously reported^{47 (link)}. 5.0 × 10⁶ purified ookinetes were incubated in 200 μL PBS at 22 °C to allow microneme protein secretion. After 6 h incubation, the supernatants were collected by a centrifugation at 750 g for 3 min, filtered through 0.45 μm filter (Millipore, cat#SLHP033RS). Equal volume of 2 × Laemmli sample buffer was added. All samples were boiled at 95 °C for 10 min and centrifuged at 12,000 g for 5 min. Equal volume of supernatants from each parasite group were used for immunoblot analysis.