Proteomics Analysis of the Nucleolus in Adenovirus-infected Cells

Lam, Yun W.; Evans, Vanessa C.; Heesom, Kate J.; Lamond, Angus I.; Matthews, David A.

2D Gel Electrophoresis-Nucleolar pellets were resuspended in 450 l of 7 M urea, 2 M thiourea, 4% CHAPS, 0.002% bromphenol blue, 0.5% (v/v) IPG buffer, pH 3-11 non-linear (GE Healthcare), and 1.2% (v/v) Destreak reagent (GE Healthcare) and loaded onto 24-cm Immobiline DryStrip gels (pH 3-11 non-linear) by passive rehydration for a minimum of 12 h. Following rehydration, the DryStrip gels were transferred to an Ettan IPGPhor 3 system (GE Healthcare), and isoelectric focusing was performed by applying 500 V for 1 h, 1000 V for 1 h, and 8000 V for 10.5 h until a total of 64,000 V-h had been achieved. Following isoelectric focusing, strips were equilibrated in SDS equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 0.002% (w/v) bromphenol blue) containing 1% (w/v) DTT for 15 min at room temperature followed by a further incubation in fresh SDS equilibration buffer containing 2.5% (w/v) iodoacetamide for 15 min at room temperature. Strips were then applied to 12.5% (w/v) SDS-PAGE gels and run at 5 mA/gel for 1 h, 8 mA/gel for an additional hour, and then at 20 watts/gel until completion using an Ettan DALT-6 separation unit (GE Healthcare). Gels were fixed for 1 h in 50% methanol and 10% acetic acid and stained overnight using SYPRO Ruby total protein stain (Invitrogen). Following destaining in 10% methanol and 7% acetic acid, the gels were imaged using a Typhoon 9400 Variable Mode Imager (GE Healthcare).