Pretreated skin was soaked in 0.5 M acetic acid with a solid to solvent ratio of 1:15 (w/v) for 48 h with continuous stirring using an overhead stirrer model W20.n (IKA-Werke GmbH & CO.KG, Stanfen, Germany). The mixtures were filtered with two layers of cheesecloth. The collagen in the filtrate was precipitated by adding NaCl to a final concentration of 2.6 M in the presence of 0.05 M tris(hydroxymethyl) aminomethane at pH 7.5. The resultant precipitate was collected by centrifugation at 20,000g at 4 C for 60 min using a refrigerated centrifuge model Avanti J-E (Beckman Coulter, Inc., Palo Alto, CA, USA). The pellet was dissolved in a minimum volume of 0.5 M acetic acid, dialysed against 25 volumes of 0.1 M acetic acid for 12 h. Thereafter, it was dialysed against 25 volumes of distilled water for 48 h. The resulting dialysate was freeze dried and referred to as ‘‘acid soluble collagen, ASC”.
Pretreated skin was soaked in 0.5 M acetic acid with a solid to solvent ratio of 1:15 (w/v) for 48 h with continuous stirring using an overhead stirrer model W20.n (IKA-Werke GmbH & CO.KG, Stanfen, Germany). The mixtures were filtered with two layers of cheesecloth. The collagen in the filtrate was precipitated by adding NaCl to a final concentration of 2.6 M in the presence of 0.05 M tris(hydroxymethyl) aminomethane at pH 7.5. The resultant precipitate was collected by centrifugation at 20,000g at 4 C for 60 min using a refrigerated centrifuge model Avanti J-E (Beckman Coulter, Inc., Palo Alto, CA, USA). The pellet was dissolved in a minimum volume of 0.5 M acetic acid, dialysed against 25 volumes of 0.1 M acetic acid for 12 h. Thereafter, it was dialysed against 25 volumes of distilled water for 48 h. The resulting dialysate was freeze dried and referred to as ‘‘acid soluble collagen, ASC”.
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