Type 1 collagen from bovine

We analyzed acid solubilized collagen derived from Rhizostoma pulmo tentacles (3.8 mg/ml in 0.1 M acetic acid), Type I collagen from rat tail (4.19 mg/ml, Millipore) and type I collagen from bovine (Sigma-Aldrich). One milligram of each sample was placed in 1.5 ml microcentrifuge tubes, freeze dried overnight, resuspended in 200 μl of 6N constant boiling HCl (Thermo Scientific) and transferred to a vacuum hydrolysis tube (Thermo Scientific). The hydrolysis tube was purged with nitrogen, evacuated and sealed. Samples were heated for 22 h at 110°C in an oven to enable hydrolysis, the vacuum was released and the HCl was evaporated by placing the open tube in an oven at 60°C for 30–40 min. The hydrosylate was resuspended in 150 μl of lithium loading buffer (Biochrom) and transferred to a 1.5 ml microcentrifuge tube. Hydrolyzed samples were transferred to glass vials and loaded in the autosampler tray after a dilution of 1:5. An injection volume of 20 ul hydrolyzed protein was analyzed for each sample. In addition, 40 μl of amino acid standard (A9906, Sigma) and 40 μl of loading buffer (a blank solution) were analyzed. Absorbance was read at 570 and 440 nm.

Type I collagen from Rhizostoma pulmo jellyfish (© 2019 Jellagen), type I collagen from rat tail (Millipore) and type I collagen from bovine (Sigma-Aldrich) were used as references.

SDS-PAGE was performed according to the method of using a 4–20% gradient. Samples were mixed with Laemmli sample buffer (Bio-Rad) with b-mercaptoethanol and heated for 5 min at 95°C. Different volumes of Rhizostoma pulmo collagen solution and 30 ug of rattail (rt) and bovine (bv) collagen were loaded to the gel and run at 100 V for 10 min followed by 120 V for 1.5 h. Following electrophoresis, protein bands were stained with Coomassie brilliant blue R- 250. Prestained-dual color marker (Biorad) was used to estimate the approximate molecular weight of collagen samples. Type I collagen from rat tail (Millipore) and type I collagen from bovine (Sigma-Aldrich) were used as references.