Gdp 3 4 3h man

Lipid-linked oligosaccharides and other glycolipids were labeled in a cell-free system as described [17 (link),18 (link)]. Briefly, washed cell lysates were supplemented with 5 mM MnCl₂ and 1 mM DTT, incubated with GDP[3,4-³H]Man (0.5 μCi/mL, 18.3 Ci/mmol; PerkinElmer Life Sciences) and 2 mM UDP-GlcNAc for 5 min. Tunicamycin (0.8 μg/mL, Boehringer Mannheim, Indianapolis IN) was added to facilitate the identification of the dolichol-linked N-glycan precursor. The reaction was continued after the addition of 1 mM GDP-Man (20 min at 27 °C) and then terminated by addition of CHCl₃/CH₃OH (1:1, v/v) to yield CHCl₃/CH₃OH/H₂O in a 10:10:3 (v/v/v) ratio. Insoluble material was removed by centrifugation and organic supernatants dried under nitrogen. Lipids were extracted by addition of equal volumes of n-butanol and water, with the organic upper phase removed to a new tube. The lower aqueous phase was re-extracted twice with water-saturated n-butanol and the organic phases pooled and dried. Samples were then analyzed on silica gel 60 TLC plates (Merck) with plates developed in CHCl₃/CH₃OH/H₂O (10:10:3, v/v/v), dried, and sprayed with En³Hance (DuPont) and exposed to film at −80 °C.