Mini protein 2 dual slab cell

Tricine–SDS-PAGE is commonly used to separate proteins in the mass range of 1–100 kDa. It is the preferred electrophoretic system for proteins with a resolution smaller than 30 kDa. A sample (5 µg or 12 µL) was mixed with 4× Laemmli sample buffer (250 mM Tris, pH 6.8; 12% glycerol; 4% sodium dodecyl sulfate (SDS); 10% beta-mercaptoethanol; 0.05% bromphenol blue) and then boiled for 5 min at 95 °C. Following the method described by Schägger and Jagow [58 (link)], samples were separated on a gel after cooling (7 × 8 cm; 4% stacking gel [4% acrylamide: bisacrylamide (29:1)]; 68 mM tris, pH 6.8; 0.2% SDS; 0.2% N,N,N′,N′-tetramethylethylenediamine (TMED); 0.03% ammonium persulphate (APS) with 18% separating gel [18% acrylamide: bisacrylamide (32:1)]; 1 M Tris, pH 8.45; 0.1% SDS; 14% glycerine; 0.05% TMED; 0.05% APS) at 150 V for 180 min (Mini-protein II Dual Slab Cell, Bio-Rad, Feldkirchen, Germany). The protein sizes were calculated by comparing the migration of the protein bands to molecular mass standards (Mark12, Thermo Darmstadt, Germany) after the gel was stained with mass spectrometry-compatible silver staining (Proteome Factory PS-2001, Proteome Factory AG, Berlin, Germany). The gel was scanned using a ScanMaker 9800 XL plus scanner (Microtek International Inc., Hsinchu, Taiwan) with a transparency adaptor at a resolution of 150 dpi.

Proteome Factory AG (Berlin, Germany) performed 1D gel electrophoresis using 12% SDS-PAGE gels under reducing conditions (according to Laemmli) [15 (link)]. Samples were mixed with 1/5 volume 5 x Laemmli sample buffer (125 mM tris, pH 6.8; 6% glycerol, 2% SDS; 5% beta-mercaptoethanol; 0.025% bromophenol blue) followed by boiling at 95°C for 5 min. After cooling, samples were separated on 15% SDS-PAGE [7x8 cm; 4% stacking gel (4% acrylamide: bisacrylamide (29:1); 68 mM tris, pH 6.8; 0.2% SDS; 0.2% tetramethylethylenediamine (TEMED); 0.03% ammonium persulfate (APS); 15% separating gel (15% acrylamide: bisacrylamide (29:1); 375 mM tris, pH 8.8; 0.1% SDS; 0.05% TEMED; 0.05% APS)] at 150 V for 75 min (Mini-protein II Dual Slab Cell, Bio Rad). Protein bands were visualized using coomassie blue or silver-stain.

The sample (5 µg up to a maximum of 12 µL) was mixed with 4× Laemmli sample buffer (250 mM tris, pH 6.8; 12% glycerol; 4% SDS; 10% beta-mercaptoethanol; 0.05% bromphenol blue), then boiled for five minutes at 95 °C. By [62 (link)], samples were separated on a gel after cooling (7 × 8 cm; 4% stacking gel consisting of 4% acrylamide:bisacrylamide (29:1); 68 mM tris, pH 6.8; 0.2% SDS; 0.2% TMED; 0.03% APS and 18% separating gel consisting of 18% acrylamide:bisacrylamide (32:1); 1M tris, pH 8.45; 0.1% SDS; 14% glycerine; 0.05% TMED; 0.05% APS) at 150 Volt for 180 min (Mini-protein II Dual Slab Cell, Bio-Rad, Shinagawa, Tokyo). The protein sizes were calculated by comparing the migration of the protein band to a molecular mass standard (Mark12, Thermo, Waltham, MA, USA) after the gel was stained with MS-compatible silver staining (Proteome Factory PS-2001, Berlin, Germany). The gel was scanned using a PowerLook 2100 XL scanner (Dallas, TX, USA) with a transparency adaptor at a resolution of 150 dpi.