Ettan ipgphor ief system

After protein labelling, samples were prepared for the isoelectric focusing (IEF) step. Strip gels of 24 cm and a linear pH range of 4–7 (GE Healthcare) were used. Strips were initially rehydrated with labelled protein samples (7 M urea, 2 M thiourea, 2% CHAPS (w/v), 0.5% IPG buffer (v/v) (GE Healthcare), 2% DTT). Strips were then processed using an Ettan IPGPhor IEF system (GE Healthcare) in a total of 35,000 Volts.h^-1 and, subsequently, reduced and alkylated for 30 min under slow agitation in Tris-HCl solution (75 mM), pH 8.8, containing 2% SDS (w/v), 29.3% glycerol (v/v), 6 M urea, 1% DTT (w/v) and 2.5% iodocetamide (w/v). The 2D- DIGE conditions were performed as described by Weiss and Görg [32 ]. Gels were immediately scanned with a FLA-9000 modular image scanner (Fujifilm Lifescience, Dusseldorf, Germany). To ensure maximum pixel intensity between 60,000 and 90,000 pixels for the three dyes, all gels were scanned at a 100 μm resolution and the photomultiplier tube (PMT) voltage was set between 500 and 700 V. As described by Agapito-Tenfen et al. [2 (link)], we performed preparative gels for each plant variety in order to extract relevant spots. These were performed with a 450 μg load of total protein pools in 24 cm gels from each variety, separately, and stained with colloidal Coomassie Brilliant Blue G-250 (MS/MS compatible).

Isoelectric focusing electrophoresis was performed with an Ettan IPGphor IEF System (GE Healthcare, U.S.). Before detection, protein samples were centrifuged for 2 min, then 100 μg of sample was dissolved in 800 μl of rehydration buffer (8 M urea, 0.02% CHAPS, 0.02 M DTT, and 0.05% IPG buffer). For first dimension electrophoresis, 18 cm solid phase pH gradient (pH 3–10) dry adhesive tape was rehydrated for 16 h at 21°C, and 20 μL of sample supernatant was added. Electrophoresis was performed for 15 h at 0-3500 V, followed by electrophoresis for 6.3 h at 3500 V. The focus adhesive tape was balanced twice in SDS balanced solution, and then agitated in a rocking bed for 2x15 min. The adhesive tape was removed for the second phase of vertical SDS-PAGE, with continuous flow at 40 mA for 40 min, and 60 mA 5 h until the leading edge of bromochlorophenol blue reached the bottom of glass plate. Following completion of electrophoresis, coomassie brilliant blue staining was carried out. The 2D gel image was analyzed by ImageMaster 2D Platinum software; analysis and adjustments included intensity correction, spot detection, and background reduction.

The first-dimension isoelectric focusing (IEF) was performed using the Ettan IPGphor IEF system (GE Healthcare, USA). A total of 75 μg of each labeled samples was applied on a 24 cm pH 3–11 NL Immobilized DryStrip (GE Healthcare). IEF was performed using the following conditions: 18 h at 50 V, 12 h at 300 V, 2 h at 500 V, 2 h at 1000 V, and 8 h at 8000 V. Each time, four or six IPG strips were run in parallel. After the first-dimension IEF, each strip was equilibrated for 15 min in the reducing equilibration buffer (6 mol/L urea, 30% (v/v) glycerol, 75 mmol/L Tris-HCl buffer (pH 8.8), 2% (w/v) SDS, and 1% (w/v) DTT) at room temperature. Subsequently, each strip was re-equilibrated in the similar buffer containing 4.5% iodoacetamide (IAA) instead of DTT for 15 min. After the equilibration, each equilibrated strip was loaded on the top of 12.5% SDS-PAGE gels (25 mM Tris, 192 mM glycine, 0.1% SDS, 0.02% bromophenol blue, 0.5% (w/v) agarose) for the second dimension. Gels were run in Ettan DALTsix Electrophoresis System (GE Healthcare), and the electrophoresis was conducted under the following conditions: 1 W/gel for 1 h and 10 W/gel for 5 h in the dark at 12°C. Gels were immediately scanned using a Typhoon Trio Variable Mode Imager (GE Healthcare).