Denaturing Co-Immunoprecipitation Protocol

Traditional co-IP was carried out as previously described [61 (link)]. For denaturing co-IP of crosslinked proteins, irradiated parasites were lysed in a 1% SDS/50 mM Tris, pH 8.0/150 mM NaCl buffer and boiled at 100°C for 10 minutes to completely denature protein complexes. The lysate was centrifuged, and the supernatant was diluted 10-fold to RIPA conditions prior to IP. Precipitated proteins are either eluted in sample buffer or by high pH with a 100 mM triethylamine solution and dried using a vacuum concentrator. Colloidal Coomassie staining was accomplished using GelCode Blue Stain (ThermoScientific). Gel slices were excised and processed for mass spectrometry. IPs were performed using rat anti-HA (Roche) or mouse anti-V5 (Sigma) agarose beads.