*3.2. Molecular Mass by SDS-PAGE and Amino Acid Profile*

The protein molecular mass analysis was done by SDS-PAGE using 10% running gel, 4% stacking gel, 20 microliter sample loading volume (10 micrograms per lane) and 120 voltage as per our earlier method [10]. After the run, the gels were stained with Coomassie blue R-250 in methanol and acetic acid for 30 min followed by detaining with methanol and acetic acid [32]. The amino acid profile was determined by hydrolyzing the samples in 6M hydrochloric acid at 110 ◦C for 24 h and then injected (aliquot of 0.4 mL) into an amino acid analyzer (Hitachi L-8800, Tokyo, Japan) and expressed as residues per 1000 residues [1].

#### *3.3. Viscosity and Solubility*

The viscosity and solubility of the collagen samples were done as per our earlier method [1]. For viscosity, the samples were heated from 5 to 40 ◦C followed by injection to a Brookfield LVDV-II+P viscometer (Brookfield Engineering Laboratories Ltd., Middleboro, MA, USA) to measure relative viscosity. The percentage of collagen solubility was tested against different pH (2–10) and salt concentrations (1–6%). The relative solubility was measured from the protein content [33].
