Establishment and optimization of two-dimensional gel electrophoresis for epidermal proteome of turbot Scophthalmus maximus

The two-dimensional gel electrophoresis for the epidermal proteome of Scophthalmus maximus was established and optimized through comparative tests by using different extraction methods, isoelectric focusing programs and sample volumes. The total proteins of the turbot epidermis were extracted by the combination of improved trichloroacetic acid(TCA)/acetone and ultrasonic extractions. The lysis buffer based on the Bio-Rad recommendations was optimized. Protein of 250 mu g was loaded on the 17cm pH 4-7 immobilized pH gradients(IPG)strips. The programs for the gel isoelectric focusing were 30V linear ramp, 6h/250V linear ramp, 2.5h/500V linear ramp, 1.5h/1000V rapid, 2h/4000V linear ramp, 3h/8000V rapid, 3h/8000V linear ramp, and 1h/500V 24h.The gel was stained with sliver nitrate, scanned and analyzed using the Image Master 2D Platinum 7.0 software. The two-dimensional gel electrophoresis analysis of the epidermal proteome of the turbot was successfully established with high quality map of separation under the