2.3.1. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The collagen was prepared as a 1 mg/mL sample solution, mixed with the 4× Laemmli Sample Buffer (1610747, Bio-Rad Laboratories, Hercules, CA, USA), and then heated at 100 °C for 3 min. An 8% separating gel and 3% stacking gel were prepared. A protein marker (26634) was used to estimate the molecular weight of the samples, and rat tail type I collagen was used as a reference. The samples were electrophoresed for 65 min at 110 V, 60 mA using an electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA). The gels were fixed with 50% (v/v) ethanol and 10% acetic acid for 30 min, stained with 0.125% (w/v) Coomassie Brilliant Blue, 50% (v/v) ethanol, and 10% acetic acid for 30 min, and decolorized using 50% methanol and 10% acetic acid for 20 min. Finally, the protein molecular weights were estimated using Quantity One software (VERSION 4.6.0, Bio-Rad Laboratories, Hercules, CA, USA), and the bands were analyzed using Image J software (VERSION 1.8.0, National Institute of Mental Health, Bethesda, MD, USA).