Pharmalyte

The frozen LM tissues (100 mg) from Shaziling pigs and Yorkshire pigs were crushed in a mortar containing liquid nitrogen and were then sonicated for 10 s using a Sonoplus (Bandelin Electronic, Berlin Germany). The crushed tissue was homogenized in 1 mL of cold dissolution buffer containing 7 M urea, 2 M thiourea, 1 % dithiothreitol (DTT), 4 % (w/v) CHAPS, 20 μL protease inhibitor cocktail (BBI, Canada), and 2 % (v/v) pharmalyte (pH 3–10; BioRad, Hercules, CA, USA). The homogenate was centrifuged at 15,000 × g, for 20 min at 4 °C. The supernatant fraction was filtered and kept at −80 °C for subsequent analysis. The total protein content was determined using a Bradford assay kit (Bio-Rad).

50 μg of total protein per sample, in triplicates, was brought to a final volume of 210 μL with rehydration buffer (5 M urea, 0.5% CHAPS, 0.5% Pharmalyte (Bio-Rad, Hercules, CA, USA), and 12 μL/mL of DeStreak reagent (GE Healthcare)) and subsequently applied on immobilized pH gradient (IPG) strips (ReadyStrip pH 3–10, nonlinear, 11 cm, Bio-Rad). The strip was covered with silicone oil, actively rehydrated (50 V, 12 h, 20°C), and then focused (Bio-Rad Protean I12) by increasing the voltage to 5000 V (total 50 kVh, current limit 30 μA/strip). Focused strips were stored at −80°C until further use. Before second dimension, each strip was incubated twice for 20 min in 2 mL equilibration buffer (6 M urea, 2% (w/v) sodium dodecyl sulfate (SDS), 25% glycerol, and 3.3% 50 mM Tris/HCl pH 8.8, stained with bromophenol blue) first supplemented with 10 mg/mL dithiothreitol (DTT) and then 48 mg/mL 2-iodoacetamide (IAA). The second dimension was carried out using precast Criterion TGX Stain-Free polyacrylamide gels (133 × 87 × 1 mm, Bio-Rad) on a Criterion cell (Bio-Rad) for 2 hours at 20 mA.