Panel A) Schematic representation of the extraction protocol for membrane proteins. Metacyclic trypomastigotes (1 × 108) were lysed in TBS containing 2% Triton X-114 (TX-114). The homogenate was clarified by centrifugation and the supernatant stored at −20 °C for 24 h. The supernatant was then submitted to phase separation with TX-114 at 37 °C. The final detergent-rich extract corresponds to the GPI-anchored protein-enriched fraction. Panel B) Analysis by SDS-PAGE and Coomassie staining of molecules obtained in the purification steps of GPI-anchored proteins. SBA, supernatant from extraction step with buffer A; P1, Pellet 1; S2, supernatant 2; S3, supernatant 3; S5, supernatant 5; P2, pellet 2; GPI, GPI-rich fraction. The relative molecular masses are indicated on the right.