Trypanosoma Glycoprotein Deglycosylation

Bloodstream form trypanosomes were cultured in the presence of tunicamycin (Sigma) added to complete media at 1μg/ml, which prevents further cell proliferation (52 (link)). For treatment with PNGase F, 1 × 10⁸ cells were washed in PBS and incubated with lysis buffer (1% NP-40, 100mM NaPO4, pH7.5) plus protease inhibitor cocktail (Roche) and then heated to 95°C for 15 minutes. Samples were treated with 10mU PNGase F (NEB) overnight at 37°C. A second aliquot of PNGase F was added and the reaction continued for 2 hours prior to analysis. For treatment with Endoglycosidase H, 1 × 10⁸ cells were washed and lysed by incubation with 1ml of 5mM EDTA and protease inhibitor cocktail (Roche), followed by two cycles of freeze-thawing. Samples were centrifuged to separate crude membranes and the pellet dissolved in 10mM Tris-HCl, pH8.0, 1mM EDTA, 1% SDS. 5mU/ml of Endoglycosidase H (Calbiochem) was added to each sample, and then 5mU/ml again 12 hours prior to analysis.