Purification and Western Blot Analysis of Plasmodium Proteins

P. falciparum parasites were released from erythrocytes by treatment with 0.15% saponin in PBS. Murine erythrocytes infected with the pv5-tag-GFP^PV or exp2-mCherry P. berghei lines (62 (link)) were purified on a Nycodenz gradient and lysed hypotonically for 1 h on ice in 10 mM Tris⋅HCl, pH 7.5. P. berghei lysates were centrifuged for 50 min at 100,000 × g. Membrane pellets were resuspended in 0.1 M Na₂CO₃ in PBS or in 1% Triton X-100 in PBS, respectively, and centrifuged for 50 min at 100,000 × g. Proteins were separated on SDS-polyacrylamide and transferred to nitrocellulose membranes. Western blotting was performed using rat anti-mCherry (1:5,000; ChromoTek), chicken anti-GFP (1:5,000; Abcam), rat anti-HA (1:1,000; Sigma Aldrich), rat anti-PfBiP (1:1,000) (63 (link)), and rabbit anti-human hemoglobin α-primary antibodies (1:1,000; Abcam) followed by chemiluminescence detection with horseradish peroxidase-coupled secondary antibodies (1:10,000; Sigma Aldrich, or 1:5,000; Jackson ImmunoResearch).