Western Blot Protocol for Low MW Proteins

Proteins from saponin-lysed schizonts or supernatant and merozoite fractions from PIA assays, were separated on 4%–12% or 3%–8% acrylamide gels (NuPAGE, Invitrogen). Separated proteins were transferred to nitrocellulose membranes by electroblotting. Blots were probed with HRP-conjugated anti-HA antibody (Roche) 1:1000 or for two-step methods, a primary antibody was followed by HRP-conjugated secondary antibody (Millipore). Bands were detected using ECL Plus Western blotting reagent (GE Healthcare) and the ChemiDoc Imaging System (Biorad). Antibodies used in this study are described in Table S4.
To ensure low MW proteins less than 20 kDa as in Fig. 1f, remained on the nitrocellulose membrane during the blocking, antibody and washing steps, the membrane was subject to a post-blot MeOH fixation protocol^{43 (link)}. Briefly, after electroblotting, the membrane was placed in 50% MeOH for 30 min/4 °C, then 50% MeOH for 30 min/50 °C, before the usual Western blot procedure.