Streptavidin horseradish peroxidase

Denatured supernatants of monomeric biotinylated proteins were resolved using NuPAGE™ Novex™ 4–12% Bis-Tris protein gels, before blotting onto nitrocellulose membrane (GE Healthcare). After blocking, membranes were incubated with streptavidin-horseradish peroxidase (1:2000, Cell Signaling Technology) for 1 h and developed with Amersham ECL Prime chemiluminescence substrate (GE Healthcare).

Supernatants from BMDMs that had been treated with LLOME for the indicated time-points were diluted in MES reaction buffer and incubated at 37 °C for 15 mins with 10 μM biotin-PEG-LVG-DMK. The reaction was terminated by addition of 5X Laemmli buffer and denaturation at 95 °C. Samples were resolved by 15 % SDS-PAGE and transferred onto nitrocellulose PVDF membranes (Millipore), blots were subsequently blocked in 5 % BSA in TBS/tween overnight at 4 °C. The membrane was then incubated with Streptavidin-horse radish peroxidase (Cell Signaling) diluted TBS/Tween, with 5 % BSA for 0.5 h at room temperature. Proteins were imaged using ECL Plus chemiluminescent substrate (ThermoFisher Scientific) and exposed using the ChemiDoc XRS imaging system (BioRad Laboratories).

P. vivax biotinylated bait proteins were reduced with NuPAGE Sample Reducing Agent (Invitrogen), heated at 70°C for 10 min, fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. After blocking in HBS/0.1% Tween/2% BSA at 4°C overnight, membranes were incubated with streptavidin-horseradish peroxidase (1:2000, Cell Signaling Technology, USA) in HBS/0.1% Tween/2% BSA at room temperature for 1–2 h, developed using the chemiluminescence substrate Amersham ECL Prime (GE Healthcare, USA), and exposed to X-ray film.