Silver stain kit

SDS-PAGE was performed in a 5, 8, 15%, or 8–15% gradient polyacrylamide gel. The gel was stained with zinc reverse staining by immersing gel in 0.2 M imidazole/0.1% SDS for 10 min, then with 0.2 M zinc sulfate. The staining reaction was quenched by rinsing with water. Alternatively, 0.25% Coomassie Brilliant Blue was used to stain protein bands.
Two-dimensional polyacrylamide gel electrophoresis (2-DE) was carried out according to the manufacturer’s protocol. 443 µg of sample was mixed with a rehydration solution (8 M urea, 0.5% 3-[(3-cholamidopropyl)dimethylanmonio]-propanesulphonic acid (CHAPS), 0.5% Bio-Lyte 3/10 (Bio-Rad, Hercules, CA, USA), 0.2% dithiothreitol, 0.002% bromophenol blue). The solution was applied to IPG strip 3–6 (BioRad). Isoelectric focusing was performed at 20 °C as follows: 12 h rehydration, 30 min at 300 V, 30 min at 1000 V, 2 h at 5000 V. After the focusing, the strip was equilibrated in 2% SDS, 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 0.25% DTT for 15 min with shaking. Then it was re-equilibrated with 2% SDS, 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 4.5% iodoacetamide for 15 min with shaking. The strip was placed on the 12% polyacrylamide gel, and second electrophoresis was performed same as SDS-PAGE. The gel was stained by using silver stain kit (Wako Pure Chemical).

Testes from normal 10-week-old mice (n = 3) were lysed by sonication in isoelectric focusing rehydration buffer (7 M urea; 2 M thiourea; 4% CHAPS; 100 mM DTT; 0.2% Bio-Lyte, pH 5–8; 0.01% bromophenol blue; and protease inhibitor). Insoluble material was pelleted (12,000 g, 15 min and 100,000 g, 60 min) and the resulting supernatant was desalted using a 2D Cleanup Kit (GE Healthcare UK Ltd, Buckinghamshire, England). One hundred microgram of protein in a total of 200 μl of rehydration buffer was applied to 24-cm pI gradient strips (pI4–7 and pI6–9; GE Healthcare UK, Buckinghamshire, England) for overnight rehydration. First-dimension isoelectric focusing was preformed on a GE Multiphor II System (GE Healthcare UK). After focusing, strips were equilibrated with equilibration buffer (6 M urea; 50 mM Tris-HCl, pH 8.0; 2% SDS; 20% glycerol; and 2% w/v DTT) for 15 min. The strips were further equilibrated with equilibration buffer II (6 M urea; 50 mM Tris-HCl, pH 8.0; 2% SDS; 20% glycerol; and 2.5% w/v iodoacetamide) for 15 min and then directly applied to a 7.5% isocratic SDS-polyacrylamide gel for the second dimension separation. The resulting gel was then transferred to a membrane for Western blot or stained using a silver stain kit (Wako Pure Chemical Industries, Osaka, Japan).