Jupiter c18 5 μm 250 4.5 mm column

Gp120 was resolved by SDS-PAGE, and the monomer excised and washed as described above. Reduction was carried out by addition of 10 mM Dithiothreitol in 100 mM ammonium bicarbonate and incubation at 65°C for 30 mins. Alkylation was subsequently performed by addition of 50 mM Iodoacetamide in 100 mM ammonium bicarbonate, with incubation at room temperature for 50 mins. Trypsin (Promega) was then added at a concentration of 12.5 μg ml⁻¹ and incubated at 37°C for 16 hours. Glycopeptides were eluted from the gel with alternate washes of water and acetonitrile, filtered through a 0.45 μM membrane (Whatman), and dried in a SpeedVac concentrator. After resuspension in 0.1% TFA, the glycopeptide pool was fractionated by reverse phase-HPLC using a Jupiter C18 5 μm 250 × 4.5 mm column (300 Å pore size) (Phenomenex) and a Dionex U3000 LC system. The following gradient was run at a flow rate of 1 ml min⁻¹, with fractions being collected every minute for 90 minutes: time = 0 min (t=0): 95% A, 5% B; t= 5: 95% A, 5% B; t=90: 10% A, 90% B; t=95: 10% A, 90% B; t=97: 95% A, 5% B; t= 120: 95% A, 5% B, where solvent A was 50 mM ammonium formate, pH 4.4, and solvent B was acetonitrile. UV absorbance was detected at 214 and 280 nm.