Tank blot device

Parasite protein samples of schizont stages (40–44 hpi) were prepared by saponin-lysis of infected erythrocytes and separated on a 12% SDS-PAGE gel at 120 V for 2 h. Separated proteins were transferred to a nitrocellulose membrane (LI-COR) for 1 h at 90 V and 4 °C using a tank blot device (Bio-Rad) according to the manufacturer’s instructions. Membranes were blocked with 5% milk in TBS followed by incubation with primary antibody diluted in 2.5% milk in TBS-T at 4 °C overnight. Primary antibodies were diluted as follows: mouse-anti-GFP (Roche) 1:2000; rabbit-anti-GFP (Thermo Scientific) 1:1000; mouse-anti-RFP (6G6, ChromoTek) 1:2000; and rabbit-anti-BiP (68 (link)) 1:2000 in 2.5% milk in TBS-T. After incubation with primary antibodies, membranes were 3× washed with TBS-T and incubated with secondary antibody for 1 h at room temperature in the dark. Secondary antibodies were diluted as follows: goat-anti-mouse800CW (LI-COR) 1:20,000; goat-anti-rabbit800CW (LI-COR) 1:20,000; goat-anti-mouse680RD (LI-COR) 1:20,000; and goat-anti-rabbit680RD (LI-COR) 1:20,000 in 2.5% milk in TBS-T. Membranes were washed again three times with TBS-T and analyzed in an Odyssey FC Imager (LI-COR).

For P. berghei parasite protein extracts, blood of infected mice was washed twice in 1 × PBS and infected red blood cells purified on a Percoll gradient59 (link) as described for P. falciparum60 . The purified infected RBCs were washed thrice with 1 × PBS, the pellet lysed in Laemmli sample buffer and the proteins separated on 10% SDS PAGE gels followed by transfer to nitrocellulose membranes (Schleicher & Schüll) in a tank blot device (BioRad). The blots were blocked in 5% milk in 1 × PBS and reacted with the following rabbit-derived antibodies: anti-PbSBP1-mid (1:200'000), anti-PbSBP1-C (1:1'000), anti-PbMAHRP1a (1/500), anti-PfSBP1 (1/1,000) and anti-PfMAHRP1 (a kind gift of Hans-Peter Beck) (1/2,500) for 2–4 h hours. After 5 washes in 1 × PBS, goat anti-rabbit horseradish peroxidase-conjugated secondary antibody, diluted 1/2,500 (Dianova), was applied for 1 h, the membrane washed, reacted with ECL (GE Healthcare) and the signal detected using X-ray films. Alternatively, secondary antibodies were anti-rabbit IgG 800CW IRDye or IgG 680LT IRDye (both 1:10'000) and an Odyssey Imaging system (Li-Cor Bioschiences) was used for detection.

Protein samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Protran) in a tankblot device (Bio-Rad) using transfer buffer (0.192 M Glycine, 0.1% SDS, 25 mM Tris) with 20% methanol. Blocking of membranes and dilutions of antibodies were done in PBS containing 5% skim milk. Washing steps were done with PBS. Primary antibodies were applied in the following dilutions: mouse anti-GFP (Roche) 1:1000; rabbit anti-GFP (Thermo) 1:2000; rat anti-mCherry, 1:1000 (Chromotek); rabbit anti-SERA5, 1:2000 (newly raised); rabbit anti-REX3, 1:2000 (newly raised); mouse anti-SBP1N, 1:2500 (newly raised); rabbit anti-aldolase, 1:4000 (newly raised); mouse anti-HSP101, 1:1000 [7 (link)]; rabbit anti-DHFR (Abcam), 1:1000; rat anti-HA (Roche) 1:4000. Horseradish peroxidase-conjugated secondary antibodies used were goat anti-rat (Dianova) and goat anti-mouse (Dianova) and diluted 1:3000 as well as donkey anti-rabbit (Dianova) 1:2500 and applied after three washes. Immunoreactions were detected by enhanced chemiluminiscence (Bio Rad/ Thermo) and detected on CEA RP NEW x-ray films (Agfa). For quantification of Western blot signals, band intensities were measured with a Chemi Doc XRS imaging system (Bio-Rad) and densitometry analyses were done with Image Lab Software 5.2 (Bio-Rad). Data are representative of three independent experiments.