*3.4. SDS-PAGE Characterization of Acid-Soluble Collagen*

Samples (1 mg/mL) were prepared in sample buffer containing 10.52% glycerol, 21% Sodium Dodecyl Sulfate (SDS) (10%), 0.63% Dithiothreitol (DTT)and 0.5 M Tris HCl (pH 6.8) and heated for 5 min at 100 ◦C. An aliquot (8 μL) of this mixture was applied to each well in 7% polyacrylamide separating gels. Gels (100 mm × 750 mm × 0.75 mm) were prepared according to the procedure of Laemmli [32] and were subjected to electrophoresis at 20 mA using a Mini-Protean II Cell (Bio-Rad, Hercules, CA, USA). Following electrophoresis, the gels were stained with 0.04% Coomassie Blue in 25% *v*/*v* ethanol and 8% *v*/*v* acetic acid for 2 h. Excess stain was removed with several washes of destaining solvent (25% *v*/*v* ethanol, 8% *v*/*v* acetic acid). Molecular weights of subunits of ASC (acid-soluble collagen) were estimated using molecular weight standards from BIO-RAD (Hercules, CA, USA) SDS-PAGE standards high range: myosin (200 kDa); β-galactosidase (116 kDa); phosphorylase B (97 kDa); bovine serum albumin (66 kDa); ovalbumin (45 kDa).

### **4. Conclusions**

This is the first study optimizing the complete process for the extraction of acid-soluble collagen by means of three variables (temperature, time and chemical treatment concentration) from the skin of the small-spotted catshark using response surface methodology. Two-stage optimizations (alkali pre-treatment and acid extraction) of the collagen extraction process should be accomplished in a species-specific approach due to the variability of collagen extracted from different species (regarding its structure and chemical differences (for example, variations in the amino acid composition)). The variables maximizing the recovery of collagen in the first stage of extraction (alkaline pre-treatment) were 4 ◦C, 2 h and 0.1 M NaOH. The variables maximizing the recovery of collagen in the second stage of extraction (acid-soluble collagen extraction stage) were 25 ◦C, 34 h and 1 M AcOH with a yield of 61.24%. The results obtained in this study might be helpful for a potential collagen extraction upscaling study.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/1/40/s1, Table S1: Experimental domains and codification of independent variables in the factorial rotable design executed to study the optimal conditions for removing proteins different of collagen from the skin of small-spotted catshark, Table S2: Experimental domains and codification of independent variables in the factorial rotable design executed to study the optimal conditions for extraction of acid soluble collagen from the skin of small-spotted catshark.

**Author Contributions:** C.G.S., J.A.V., R.I.P.-M. and M.B. conceived and designed the experiments; M.B. performed the experiments; C.G.S., J.A.V., R.I.P.-M. and M.B. analyzed the data; M.B. wrote the paper. J.A.V. participated in the redaction of the manuscript. C.G.S., J.A.V. and R.I.P.-M. critically revised the manuscript.

**Funding:** This research was funded by EU INTERREG\_POCTEP 2015, 0302\_CVMAR\_I\_1\_P; EU-INTERREG Atlantic Area Programme, EAPA\_151/2016; Xunta de Galicia, IN607B 2018/19).

**Acknowledgments:** Authors thanks the financial support received from the projects: CVMar+i (0302\_CVMAR\_I\_1\_P, EU INTERREG\_POCTEP 2015); BlueHuman (EAPA\_151/2016, EU-INTERREG Atlantic Area Programme) and Grupos de Potencial Crecimiento (GAIN, Xunta de Galicia, IN607B 2018/19). The authors are also grateful to Helena Pazó Malvido and Marta Pérez Testa for her technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

1. Blanco, M. Valorización de Descartes y Subproductos de Pintarroja (*Scyliorhinus canicula*). Ph.D. Thesis, Universidad de Vigo, Vigo, Spain, 2015.


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