Horseradish peroxidase conjugated anti mouse igg secondary antibody

Synchronous cultures containing mainly ∼40-hpi schizonts were purified by magnetic isolation, divided into pellets of approximately 2.5 × 10⁶ schizonts, and stored frozen at −80°C. After thawing, proteins were denaturized in SDS-PAGE protein loading buffer for 5 min at 95°C and resolved by SDS-PAGE on 4 to 12% bis-Tris Criterion XT precast gels (Bio-Rad), transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked for at least 1 h in 1% casein blocking buffer (Sigma). Membranes were incubated overnight at 4°C with the following primary antibodies: purified mouse antiserum against a PfMC-2TM protein (PF3D7_0114100) at 1:200 (kindly provided by Catherine Braun-Breton) (88 (link)) and mouse anti-HSP70 antibody (89 (link)) at 1:2,000. Membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Promega) at 1:10,000, and peroxidase was detected using the Pierce chemiluminescence system (Pierce) following the manufacturer’s instructions. To control for equal loading, parts of the membranes corresponding to different molecular weight ranges were separately hybridized with different antibodies. Signal quantification was performed using ImageJ software.