*2.2. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)*

The obtained extracts were submitted to electrophoretic analyses to better characterize their biochemical composition. Conventional collagen extracts and most of proteins are easily stained with Coomassie dye also at rather low concentrations. However, this was not the case of *C. reniformis* collagens, which were detected only after staining the gels with alcian blue (Figure 2).

**Figure 2.** SDS-PAGE of ectosome (Ec) extract after one month (lane 1A, 1B) or two months (lane 2A, 2B) in DS stained with Coomassie R-250 (1A, 2A) or alcian blue (1B, 2B). A volume corresponding to 250 μg of material was added to each well.

Indeed, at 1 mg/mL, a concentration generally sufficient to well resolve the characteristic chain composition of fibrillar collagens, sponge collagen bands were rather evanescent when stained with R-250 Coomassie, but well visible when stained with alcian blue.

Comparing the protein profile one month after the start of the incubation (Figure 2—compare lanes 1A, 1B) and at the end of the second month (Figure 2—compare lanes 2A, 2B) in the DS, it is possible to see the effects of a prolonged incubation. Most of the collagen after one month in the DS is still in the form of large fibrils that cannot be solubilized and thus remain trapped in the stacking gel. However, after an additional month in DS, collagen fibrils are almost completely solubilized in their single components. In our gels we observed at least seven bands in the high molecular weight region plus a broad band that runs faster than the frontline. While some bands did not change over time (i.e., at one and two months of incubation), others appeared only after the complete incubation in DS solution, suggesting a significant increase in the solubility of collagen fibrils. Indeed, the fibrils trapped in the stacking gel (Figure 2—lane 1B), which are likely associated in small bundles as a result of interactions with other proteins, completely disappear between month 1 and 2 (Figure 2—lane 2B). On the other hand, we found a new band that was blocked at the beginning of the stacking gel. Considering its very high molecular weight, together with results detailed in Section 2.3 (below), i.e., the sensitivity to both Coomassie and alcian blue dyes as well as its resistance to a long pepsin digestion, this band was likely to have originated from the presence of isolated collagen fibrils.
