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

SDS-PAGE was performed according to the method of Laemmli (1970) [34] with a slight modification. Collagen samples (ASC and PSC) lyophilized at 0.5% with 0.1 M acetic acid were mixed with a sample plug (0.5 M Tris-HCl pH 6.8, glycerol, 10% SDS, 1% bromophenol blue, 2-mercaptoethanol, and distilled water) in a 1:1 ratio, heated to 95 °C for 60 s and allowed to cool to room temperature, the standard protein (collagen type I calf skin) was diluted 30 mg in 1 mL of distilled water, then 20 uL of the samples was loaded together with the polyacrylamide gel samples, 7.5% polyacrylamide gel (1.5 M Tris-HCl pH 8.8, 10% SDS, 30% acrylamide/bisacrylamide, 10% APS, TEMED, and distilled water) and 4% concentrating gel (0.5 M Tris-HCl pH 6.8, SDS 10%, acrylamide/bisacrylamide 30%, APS 10%, TEMED, and distilled water). For the running of the bands, an electrophoresis buffer was used (tris base, glycine, SDS, and distilled water), a voltage of 65 V for 6 h, after which time the gels were removed from the chamber (Mini-Protean tetra cell, Bio-Rad) to be stained with brilliant blue of Chromasie R-2550 at 1% (w/v), acetic acid, and methanol for 5 h, and then to be decolorized with a solution of 96% ethanol and 99.8% acetic acid for 6 h under gentle agitation. Molecular weight determination was performed on the Chemi-doc gel documentator.