Donkey anti goat igg

Normal human serum (Euro Diagnostica) samples were taken at the start of an experiment (time 0) or following 1h incubation at 37°C in the presence or absence of parasites (~50 pairs in 150 μl). Aliquots of these sera (0.5 μl) were subjected to SDS-PAGE, blotted to polyvinylidene difluoride (PVDF) membrane and blocked using 5% skim milk in phosphate buffered saline (PBS) containing 0.1% Tween 20 (PBST) for 1 h at room temperature. The membrane was then probed overnight at 4°C with one of the following polyclonal antisera: rabbit anti-human C3 (Abcam, ab117244) or goat anti-human C4 (AbD Serotec (BioRad) AHP1753) or goat anti-human factor B (Quidel A311) antiserum, all at 1:1000 dilution. Blots were washed 3 times with PBST and incubated with either donkey anti-rabbit IgG or donkey anti-goat IgG (as appropriate), conjugated to horse radish peroxidase (GE Healthcare, Pittsburgh, PA USA), at 1:5,000 dilution for 1 hour at 37°C. Following a further 3 washes in PBST, protein bands were visualized using ECL Western Blotting Detection Reagents (GE Healthcare) in a ChemiDoc Touch Imaging System (BioRad).