Goat anti rat hrp conjugate

Proteins were run on a 4–12% Bis–Tris gel (ThermoFisher) in MES buffer and then transferred to a nitrocellulose membrane. After blocking with 5% milk in PBS-T for 30 min, membranes were incubated overnight with the primary antibody in 5% milk. Primary antibodies used were: 1 μg/mL rat α HA (Roche 3F10), 1/2000 rabbit α GFP (Abcam ab290), 1/1000 rabbit α aldolase HRP (Abcam ab38905), or 1/3000 mouse α H3 (Abcam ab10799). The next day, membranes were washed three times with PBS-T and incubated for 2 h with the appropriate secondary antibody, then washed again. Secondary antibodies used were: 1/3000 goat anti-rat HRP conjugate (Millipore), 1/10,000 goat anti-rabbit HRP conjugate (Millipore), or 1/5000 goat anti-mouse HRP conjugate (Pierce). ECL reagent (Pierce) was used to detect bound antibody. The uncropped original images for all Western blots shown are provided as a Source Data file.

Full parasite protein western blot samples were collected by lysing RBCs with 0.1% saponin and boiling protein in Loading Buffer (50mM Tris-Cl pH 8.0, 20% SDS, 1% Bromophenol Blue). Fractionated parasite protein was prepared as described in [95 (link)]. Blots were performed as described [19 (link)]. Primary antibodies used were: 1/1000 rat ant-HA (Roche 3F10), 1/1000 mouse anti-GFP (Roche), 1/3000 rabbit anti-aldolase conjugated to HRP (Abcam ab38905), or 1/3000 mouse anti-H3 (Abcam ab10799). Secondary antibody concentrations used were 1/3000 goat anti-rat HRP conjugate (Millipore), 1/3000 goat anti-mouse HRP conjugate, or 1/10,000 (Pierce) goat anti-rabbit HRP conjugate (Millipore). ECL reagent (Pierce) was used to detect HRP signal. Blots were exposed to autoradiography film (VWR) and visualized using an autoradiography developer.