Representative immunoblot showing native AP65 binds to fixed parasites. Control, untreated T. vaginalis (lane 1) and trypsin-treated parasites (lane 2) were fixed and incubated with detergent lysate of T. vaginalis isolate T016 (extract) for 2 h at RT. After extensive washing, bound protein was released from the fixed organisms by boiling in electrophoresis detergent buffer  and subjected to SDS-PAGE in an 8% acrylamide gel prior to blotting onto nitrocellulose. Blots were probed with mAb DM116 to AP65. Exogenous AP65 bound to control, untreated parasites (lane 1) but not trypsin-treated organisms (lane 2). As a control, fixed parasites were also subjected to boiling in detergent and handled identically to show no AP65 reactive protein was released under the same conditions (lane 3).