Preparation of collagen
Extraction of acid-soluble collagen (ASC)
Duplicate samples were washed in distilled water at 4°C with
continuous stirring for 3 days, and the flesh cut into small pieces
and soaked in 0.5 M acetic acid with a solid/solvent ratio of 1:15
(w/v) at 4°C for a further 3 days. The mixture was filtered through
two layers of cheesecloth and the residue re-extracted under the
same conditions. The two filtrates were combined. Collagen was
precipitated by adding 0.9M NaCl with 0.05M Tris (hydroxymethyl)
aminomethane buffer, pH 7.0. The resultant precipitate was
collected by centrifugation at 20,000xg for 60 min, then dissolved in
a minimal volume of 0.5 M acetic acid and dialyzed against 50
volumes of 0.1 M acetic acid for 3 days, followed by the dialysis
against the same volume of distilled water for another 3 days. The
dialysate was freeze-dried and is referred to as acid-soluble
collagen (ASC). The yield of ASC was calculated from the dry
weight of collagen extraction in comparison with the wet weight of
the initial fresh squid used.