Quantifying PfRipr's Erythrocyte Interactome

Interaction between PfRipr and 13 erythrocyte surface proteins was quantified by AlphaScreen as reported^{40 (link)}. Briefly, reactions were carried out in 20 µl of reaction volume per well in 384-well OptiPlate microtiter plates (PerkinElmer). First, affinity-purified Ecto-PfRipr recombinant protein was biotinylated using a Biotin Labeling Kit-NH₂ (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s instruction. Secondly, 5 µl of 10 nM biotinylated protein was mixed with 5 µl of 10 nM for each erythrocyte surface protein in reaction buffer (100 mM Tris-HCL [pH 8.0], 0.01% [v/v] Tween-20 and 0.1 mg/ml [w/v] bovine serum albumin), and incubated for 1 h at 26 °C to form a protein-protein complex. Subsequently, a 10 µl suspension of streptavidin-coated donor-beads and anti-GST acceptor-beads (PerkinElmer) mixture in 1:1 (v/v) in the reaction buffer was added to a final concentration of 15 µg/ml of both beads. The mixture was incubated at 26 °C for 12 h in the dark to allow the donor- and acceptor-beads to optimally bind to biotin and GST, respectively. Upon illumination of this complex, a luminescence signal at 620 nm was detected by the EnVision plate reader (PerkinElmer) and the results were expressed as AlphaScreen counts. GST tagged Rh5, known to interact with PfRipr, was included as a positive control and His-GST as a negative control.