SDS-PAGE Analysis of Parasite Proteins

For SDS-PAGE, total protein lysates were prepared using saponin-lysed parasites resuspended with 1X Laemmli loading buffer diluted in 1x PBS supplemented with 1X Roche Complete protease inhibitors cocktail. Protein samples were separated in 4–15% polyacrylamide gels using Precision Plus Protein Standards as MW ladder (Bio-Rad) and transferred to 0.2 μm Immobilion-P^SQ transfer membrane (Millipore, Cat. No ISEQ00010) using a Bio-Rad transfer system. Membranes were blocked in 5% skim milk/1x TBS-Tween20 for 1 hour at RT. Primary and secondary antibodies were prepared in 3% skim milk/1x TBS-Tween20 and incubated for 1 hour at RT. Membranes were washed four times with 1x TBS-Tween20 for 10 min, after primary and secondary antibody incubations. The following primary antibodies were used in this study: Anti-Ty1 BB2 mouse (1:2,500; Invitrogen Cat. N^o MA5–23513), anti-PhIL1 rabbit (1:5,000 (46 (link))), anti-PfHsp70 rabbit (1:5,000; StreesMarq Biosciences Cat. N^o SPC-186D), anti-Histone 4 rabbit (1:2,000; Diagenode Cat. N^o C15410156–50). HRP-conjugated anti-mouse and anti-rabbit antibodies were used (1:5,000, Millipore). Immunoblots were incubated with the chemiluminescent substrate SuperSignal West Pico PLUS (ThermoFisher, Cat. N^o 34578) following manufacturer directions. Chemiluminescent images were obtained using an Azure c300 digital imaging system (Azure Biosystems).