Dual color protein standard 3

Directly after IEF, proteins were reduced in sample buffer (150 mM Tris-HCl, pH 8.5, 2% (w/v) lithium dodecyl sulfate (LDS), 10% (v/v) glycerol, 0.51 mM EDTA, 0.22 mM Serva Blue G250, 0.175 mM Phenol Red) containing 50 mM DTT and subsequently alkylated in sample buffer containing 125 mM iodoacetamide (IAA) for 15 min each. For protein separation in the second dimension, IPG strips were placed on precast NuPAGE Novex 4%–12% Bis-Tris ZOOM Protein Gels, 1.0 mm, IPG-well (Thermo Fisher Scientific, Waltham, MA USA), 4 µL Dual Color Protein Standard III (Serva) was used as molecular weight marker and electrophoresis was performed in MES buffer at 180 V. Gels were fixed in 40% (v/v) ethanol, 10% (v/v) acetic acid for 60 min, rinsed 3× for 10 min in water and stained with colloidal Coomassie staining solution (0.08% (w/v) Coomassie Brilliant Blue G-250, 1.6% (w/v) ortho-phosphoric acid, 8% (w/v) ammonium sulfate and 10% (v/v) methanol) over night. Excess dye was removed by washing in water. Gels were imaged using an Odyssey near-infrared scanner (LI-COR, Lincoln, NE, USA) at 700 nm wave length, intensity set to 5.0, quality set to “high” and resolution to 84 µm.