Western Blot Analysis of Malaria Proteins

Western blots of membrane proteins from pigmented trophozoite-IEs were performed using Triton-X 100-insoluble, SDS-soluble protein extracts of pigmented trophozoite-IEs as previously described [15 ]. Nitrocellulose membranes (Invitrogen) were probed with affinity-purified rat antibodies against recombinant RIF29 (1/2000) [74 (link), 75 (link)], rat antibodies against RIF40.2 (1/2000) [30 (link)], affinity-purified mouse antibodies against recombinant STEVOR3 protein (1/2000) [76 (link)], rabbit anti-SBP1 (1/1000) [20 (link)], rabbit anti-HSP70 antibodies (1/500; provided by Paul Gilson) and rabbit anti-aldolase (1/5000; provided by Jake Baum). Magnet-purified IEs were incubated with trypsin at 1 mg/mL (TPCK-treated, Sigma–Aldrich) or PBS as a negative control for 30 min at 37 °C, and the reaction stopped with trypsin inhibitor (Sigma–Aldrich) before the cells were extracted with SDS loading buffer, as previously described [30 (link)].