Affinity Purification of HA-Tagged Proteins

As described previously (22 (link)), freshly lysed parasites were harvested and incubated for 10 min in PBS with 5 mM DSSO. Tris buffer was added to a final concentration of 20 mM to quench the reaction at room temperature for 5 min. After washes, the parasites were lysed with 1 mL RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail at 4°C for 1 h. The lysate was then centrifuged, and the supernatant was incubated with 25 μL of mouse IgG magnetic beads (Cell Signaling) for 1 h at room temperature for precleaning. The unbound lysate was separated from the IgG beads and was incubated with 25 μL of anti-HA magnetic beads (Fisher Scientific) for another hour at room temperature. The anti-HA magnetic beads were washed with RIPA lysis buffer and PBS and then submitted to the Indiana University School of Medicine Proteomics Core facility for mass spectrometry.