7 cm ipg strip pi 4 7

A total of 70 μg of dissolved protein sample was separated by 2DE in the first dimension by isoelectric focusing on a 7 cm IPG strip (pI 4–7) (GE Healthcare, UK) and the second dimension by SDS-PAGE on a Protean II unit (Bio-Rad Hercules, USA), according to methods given by Muneer et al. [25 (link)]. The samples were rehydrated for 12 h (with 125 μL rehydration buffer containing 70 μg proteins) before focusing. For the first dimension, the rehydrated strips were focused at 20°C with 50 μA current per strip using a four-step program: step and hold -300 V for 30 min, gradient, -1000 V for 30 min, gradient, -5000 V for 1 h 30 min, and final step and hold 1-2 h until the final voltage reached 10000 V. The focused strips were equilibrated twice for 15 min in 10 mg·mL⁻¹ DTT and then in 40 mg·mL⁻¹ iodoacetamide prepared in equilibration buffer containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, and 2% (w/v) SDS. After equilibration, strips were attached to the second dimension gel (12.5%) with 0.5% low melting point agarose sealing solution. Electrophoresis was done at a constant voltage of 80 V for 4 h until the bromophenol dye front reached the end of the gel. The protein spots in the analytical gels were stained using a silver staining method [26 (link)].

A total of 70 μg of dissolved protein sample was separated by the 2-DE in the first dimension by the isoelectric focusing on a 7-cm IPG strip (pI 4–7) (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) and the second dimension by SDS-PAGE on a Protean II unit (Bio-Rad, Hercules, CA, United States), according to methods given by Muneer et al. (2015) (link). The samples were rehydrated for 12 h (with a 125-μl rehydration buffer containing 70 μg proteins) before focusing. For the first dimension, the rehydrated strips were focused at 20°C with 50 μA current per strip using a four-step program: step and hold—300 V for 30 min, gradient—1,000 V for 30 min, gradient—5,000 V for 1 h 30 min, and final step and hold for 1–2 h until the final voltage reached 10,000 V. The focused strips were equilibrated twice for 15 min in 10 mg ml^–1 DTT and then in 40 mg ml^–1 iodoacetamide prepared in an equilibration buffer containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, and 2% (w/v) SDS. After equilibration, the strips were attached to the second dimension gel (12.5%) with a 0.5% low melting point agarose sealing solution. Electrophoresis was done at a constant voltage of 80 V for 4 h until the bromophenol dye front reached the end of the gel. The protein spots in the analytical gels were stained using the silver staining method.