Immunoblots were performed using saponin-lysed, infected erythrocytes. Parasite proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) as described previously (63 (link), 64 (link)) and transferred to a nitrocellulose membrane (Amersham Protran; 0.45-μm-pore-size nitrocellulose membrane; GE Healthcare) using a Trans-Blot device (Bio-Rad) according to the manufacturer’s instructions. The membranes were blocked with 3% skim milk in Tris-buffered saline (TBS) for 30 min and then probed with mouse anti-GFP (clone 7.1 and 13.1; 1:1,000, Roche) or rabbit anti-aldolase (65 (link)) (1:2,000). The chemiluminescent signal of the horseradish peroxidase-coupled secondary antibodies (Dianova) was visualized using a Chemi Doc XRS imaging system (Bio-Rad) and processed with Image Lab 5.2 software (Bio-Rad).
To perform loading controls and ensure equal loading of parasite material, rabbit antialdolase (65 (link)) antibodies were used. The corresponding immunoblots were incubated twice in stripping buffer (0.2 M glycine, 50 mM dithiothreitol, 0.05% Tween 20) at 55°C for 1 h and washed three times with Tris-buffered saline for 10 min before reprobing.
To perform loading controls and ensure equal loading of parasite material, rabbit antialdolase (65 (link)) antibodies were used. The corresponding immunoblots were incubated twice in stripping buffer (0.2 M glycine, 50 mM dithiothreitol, 0.05% Tween 20) at 55°C for 1 h and washed three times with Tris-buffered saline for 10 min before reprobing.
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