2.5.4. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Protein patterns of surimi gels and pastes were analyzed by SDS-PAGE following the method of Singh and Benjakul [26]. Samples (2 g) were firstly mixed with 5% (w/v) SDS solution (85 °C) and homogenized using a homogenizer (IKA Labortechnik, Selangor, Malaysia) at 10,000 rpm for 1.5 min. The mixture sample was heated for 1 h at 85 °C. The samples were centrifuged at 7000× g for 15 min and afterward, the protein content of each sample was analyzed by the Biuret method using bovine serum albumin as the standard [27]. The protein content of each sample was then adjusted to 6 mg/mL. Subsequently, samples were mixed with sample buffer containing 10% (w/v) β-mercaptoethanol, 4% (w/v) SDS, 20% (w/v) glycerol, and 0.001% (w/v) bromophenol blue at a ratio of 1:1 (v/v) and boiled at 85 °C for 30 min before being loaded. The polyacrylamide gels were prepared using 7.50 or 10% (w/v) running gels and 4% (w/v) stacking gel. Samples (15 µg protein) were loaded and subjected to electrophoresis at a 15 mA current per gel, using a Mini Protein II unit (Bio-Rad Laboratories, Inc., Richmond, CA, USA). Standard markers were also used. After separation, the proteins were stained for 24 h using 0.05% (w/v) Coomassie Blue R-250 in 50% (v/v) methanol and 7.5% (v/v) acetic acid, followed by de-staining using the mixture of 30% (v/v) methanol and 10% (v/v) acetic acid for 6 h. Band intensity of myosin heavy chain (MHC) with a molecular weight around 200 kDa of surimi paste and gel appearing in 7.50 or 10% (w/v) running gel was determined by an image analysis using a Model GS-700 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA, USA) with the aid of molecular Analyst Software version 1.4.