Anti rabbit horseradish peroxidase conjugated secondary antibody

Detection of the GFP antigen in HEK 293 and Oli-neu protein lysates following sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotting was performed as described (Harasta et al., 2015 (link)). Forty eight hours (HEK 293) and 96 h (Oli-neu) post transfection, protein lysates were extracted and 10 μg of total protein was size separated and immobilized on a polyvinyl fluoride membrane. Equal loading was confirmed by staining the membrane with Ponceau S. After washing and blocking using 5% milk powder, the membranes were incubated with a rabbit anti-GFP antibody, produced in-house (von Jonquieres et al., 2013 (link)). This was followed by application of an anti-rabbit horseradish peroxidase-conjugated secondary antibody (Dianova, Hamburg, Germany), detection of the immunoreactivity by the enhanced chemiluminescence system (BioRad, Gladesville, NSW, Australia) and signal capture (GelDoc, BioRad, Gladesville, NSW, Australia).

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.