Supersignal west femto chemiluminescent kit

To determine expression of Atg8 in ring, trophozoite, and schizont stages, parasite pellets were suspended in 10 mM Tris, pH 7.5 and equal volume of 2× SDS-PAGE sample buffer (1× buffer contains 62.5 mM Tris-HCl, 20% glycerol, 2% SDS, 1% β-mercapto ethanol, 0.01% bromophenol blue, pH 6.8) was added to the lysate, and the sample was sonicated (3 pulses of 15 seconds each at 20% amplitude). The sample was incubated at 100°C for 10 min, centrifuged at 24000×g for 20 min, and supernatants were transferred to fresh tubes and used for western blot. The supernatants of parasite lysates (corresponding to approximately 1×10⁸ parasites) were run on a 10% SDS-PAGE gels, and transferred onto the Immobilon-P membrane. The membranes were treated with 1% paraformaldehyde [82] (link), blocked with blocking buffer (5% milk in PBS-Tween 20) for two hours at room temperature, incubated with primary antibodies (rabbit anti-Atg8 at 1/1000, rabbit anti-Hsp70 and mouse anti-β-actin at 1/500), in blocking buffer for overnight at 4°C. The blots were washed with the blocking buffer, incubated with secondary antibodies (HRP-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse IgGs were used at a dilution of 1/10,000 in blocking buffer) for 1 hour at room temperature, and signal was developed with the SuperSignal West Femto Chemiluminescent kit (Thermo Fisher Scientific) on X-ray film.