Hrp conjugated goat anti rat igg

To obtain total parasite protein extracts, infected erythrocytes were lysed with cold 0.1% saponin and washed with cold PBS containing protease inhibitors (Roche #11873580001). Isolated parasites were directly resuspended in SDS-PAGE loading buffer, boiled for 5 min at 95ºC and stored at −80ºC. Before performing SDS-PAGE, β-mercaphotetanol was added to a final concentration of 4% and samples boiled again for 5 min at 95ºC. Protein extracts were separated on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes following standard procedures. We used a rat anti-HA monoclonal antibody (Roche #11867423001) at 1:200 and as a secondary antibody an HRP conjugated goat-anti-rat IgG (Thermo Fisher #A10549) at 1:1,000. To control for equal loading of parasite material between different samples, membranes were stripped with Restore stripping buffer (Thermo Scientific) according to the manufacturer’s instructions and re-probed with an antibody against PfHSP70 (1:10,000; StressMarq Biosciences #SPC-186C, lot #1007) and an HRP conjugated goat-anti-rabbit IgG (Sigma #A6154) secondary antibody at 1:5,000.