3.4.1. Sodium Dodecyl Sulfate Gel Electrophoresis

The determination of the collagen's electrophoretic profiles was performed according to Laemmli [82] method. The extracted collagens ASC and PSC (1 mg/mL) were dissolved in 0.02 M sodium phosphate buffer (pH 7.2) containing urea (3.5 M) and sodium dodecyl sulfate (SDS = 1% *w*/*v*). The solubilised samples were then mixed with a Tris HCl buffer (0.5 M, pH 6.8) containing 10% (*w*/*v*) SDS, 50% (*v*/*v*) glycerol and 5% (*v*/*v*) b-mercaptoethanol (b-ME), at 1:1 (*v*/*v*) ratio. Electrophoresis was carried on a polyacrylamide gel made of 7.5% running gel and 4% stacking gel. Following 150 min of electrophoretic migration, the protein bands were stained with Coomassie brilliant blue R-250 (0.1%) in methanol and acetic acid (45%, 10% *v*/*v* respectively). After that the gel was finally distained with methanol and acetic acid (10%, 10% *v*/*v* respectively). High molecular weight markers (Biorad, CA, USA) were used to estimate collagen molecular weights.

#### 3.4.2. Peptide Mapping

The ASC and PSC peptide mappings were determined according to the method of Kittiphattanabawon et al. [83] with a slight modification. To solubilise collagen, samples (6 mg) were suspended in 0.1 M sodium phosphate buffer (pH 7.2) containing 0.5% SDS, then heated at 100 ◦C for 5 min. The digestion of collagen in solutions (300 μL) was realized by adding 200 μL of Lysyl endopeptidase (from *Achromobacter lyticus*, 10 μg/mL buffer) and incubating at 37 ◦C for 5 min

and 25 min. The proteolysis was stopped by incubating samples in boiling water for 3 min, then SDS-PAGE was realized using 12% running gel and 4% stacking gel.

**Figure 9.** Collagen extraction process from smooth hound skin (ShS).
