Ipg strips with 4 7ph range

Total cell lysates were prepared in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% NaDoc, 0.1% SDS, 50 mM Tris pH 8) containing protease and phosphatase inhibitors. Protein concentration was measured using bovine serum albumin as a standard^{58 (link)}. Isoelectric focusing (IEF) was done using the Protean IEF Cell (Bio-Rad) at 20°C with rapid ramping to voltage 10,000 V at a current limit of 50 μA using IPG Strips with 4–7pH range (Bio-Rad). Where indicated total cellular fractions were treated with λ-phosphatase according to manufacturer’s instructions (New England BioLabs) before isoelectric focusing. Phos-tag PAGE was performed according to manufacturer’s instructions (Wako) using 100 μM Phos-tag. Samples were subjected to SDS-PAGE, transferred to nitrocellulose membrane, blocked with low-fat milk and incubated with primary antibody overnight. The proteins were visualized by using peroxidase-conjugated secondary antibodies and chemiluminescent reagent (PerkinElmer) in LAS-3000 Imaging System (Fujifilm). Densitometric quantification was performed on unsaturated images using ImageJ.