Zoom nupage gel

Co-immunoprecipitation was performed as in Mazzotta et al. (2013 (link)). The 2D electrophoresis was performed according to Khoudoli et al. (2004 (link)), with some modifications. Protein complexes were solubilized by heat treatment (5 min at 95°C) in presence of 100 mM DTT and 0.2% SDS, precipitated in 80% acetone at −20°C and solubilized for 6 h in resuspension buffer (30 mM Tris Base, 7 M Urea, 2 M Thiourea, 1.2% CHAPS, 0.14% ASB14, 0.25% Ampholytes, 43 mM DTT), with the addition of 60 mM Acrylamide after 3 h, in order to alkylate the proteins (Mineki et al., 2002 (link)). Isoelectric focusing (IEF) was performed in 7 cm IPG strips of pH range 4–7 (ReadyStrip™_Bio-rad); strips have been passively rehydrated for 16 h and then iso-electro focused by a two-phase protocol: 30 min at 250 V, 3 h and 30 min at 5500 V and 500 V until the complete focusing. After IEF, strips were equilibrated in Equilibration buffer (50 mM Bis-Tris pH 6.4, 6 M Urea, 30% (w/v) glycerol, 2% SDS) containing 50 mM DTT for 20 min and 360 mM Acrylamide for further 20 min. Strips were then placed on a 4%–12% pre-cast “ZOOM NuPAGE gel” (Invitrogen^®) with the help of a 0.5% agarose matrix and run at room temperature at 50 V.

Cells were lysed in IEF lysis buffer (8 M urea, 1% NP-40, 10 mM DTT) with fresh protease and phosphatase inhibitors (Sigma-Aldrich). Proteins were separated by isoelectric focusing using the Ettan IPGphor II isoelectric focusing system (GE) and separated by molecular weight using 4–12% Bis-Tris ZOOM NuPAGE gel (Invitrogen). A 7 cm Immobiline DryStrip (pH 3–10) was rehydrated in 0.5% IPG DeStreak rehydration buffer for 24 h (GE). Two hundred micrograms of whole cell lysate were mixed with DeStreak buffer and loaded on a Immobiline DryStrip, and IEF was performed according to manufacturer’s instructions. Following IEF, the strip was equilibrated in 1X LDS loading buffer with 1X DTT (Invitrogen) and loaded into the single well of the ZOOM NuPAGE gel and the standard Western protocol was followed.