Nile Tilapia Case Analysis

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2.1. Fish Scales Preparation and Demineralization:
Fish Scales of Nile Tilapia, caught from Nile Delta, were randomly collected from the local market during the month of June. The scales were washed thoroughly with distilled water, and stored at –20°C until used. All reagents used were of analytical grade. Fish scales were washed twice in 10 wt.% of NaCl solutions (scales: solution = 1:10) to remove non-collagenous proteins on the surface by stirring the solution for 24h, then washed thoroughly with distilled water until the pH was neutral. Demineralization was achieved by stirring scales for 90 min. in 0.4 mol/l HCl (scales: solution = 1:15), followed by washing the scales for three times in distilled water to be ready for collagen extraction.
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The collagen samples were dissolved in 0.1 M acetic acid with different dilutions. The dissolved collagen samples were mixed with the sample buffer (0.5 M Tris-HCl buffer pH 6.8 which contained 30% (v/v) glycerol, 5% (w/v) SDS, 10% (v/v) β-mercaptoethanol, and 0.04% (w/v) Bromophenol blue). Coo- massie Brilliant Blue G-250 was used to visualize the gel after the electrophoresis. High molecular weight protein markers were used to estimate the molecular weight of proteins.

2.3.2 Amino Acid Analysis:
The amino acid composition of collagen was analyzed with high performance amino acid analyzer (Biochrom 30, Biochrom Limited, Cambridge, England). Lyophilized samples were dissolved in 6 M HCl solutions and hydrolyzed at 110°C for 24 h.
Hydroxyproline content was detected using The GC/MS QqQ system (Agilent 7890A series GC system coupled with an Agilent 7000B QqQ MS, Agilent Technologies UK Ltd). Derivatization has been done according to the protocol developed by Villas-Boas et al. [23]. A Standard spectrum was first done using hydroxyproline standard, to determine the mass spectrum of hydroxyproline, which was later used to quantify hydroxyproline.
2.3.3 Determination of Denaturation Temperature
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BHK-21 cells were seeded on the 24-well collagen gel coated plate, containing Hank 's Balanced Salt Solution (HBSS) with Phenol Red (BioWhittaker™, Lonza, Belgium), supplemented with 10% fetal bovine serum (FBS) (Gibco Invitrogen, Milan, Italy), with 1% penicillin/ streptomycin (Sigma-Aldrich, Inc., St. Louis, USA), at density of 3×104 cells per well. Some wells were kept untreated as controls to compare with tests. The medium was changed every 2 days. At days 1 and 2, the cells on the collagen gels were observed by an inverted light microscope (DMI3000 B; Leica,

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