Mini protean 2 cell system

The yield of collagen extracted was calculated as a percentage of the lyophilized collagen obtained from skin and bone biomass on a dry and wet basis.
SDS–polyacrylamide gel electrophoresis for the characterization of collagen fractions was performed according to Sotelo et al. (2016 ) with some modifications. Lyophilized collagen was dissolved in sample buffer (0.5 M Tris-HCl at pH 6.8, 10% glycerol, 2% SDS, 0.6% DTT, 0.026% bromophenol blue) at a concentration of 1 mg/ml (w/v) and heated at 100 °C for 4 min. A total of 8 µl of the treated sample was loaded per well in a 7% acrylamide—0.24% bis-acrylamide separating gel (100 × 750 × 0.75 mm) and subjected to electrophoresis at a constant current of 15 mA using a Mini-Protean II Cell system (Bio-Rad Laboratories, Hercules, CA, USA). Then, the gels were stained with 0.04% Coomassie Blue in 25% v/v ethanol and 8% v/v acetic acid for 30 min at 60 °C. Excess stain was removed with several washes of destaining solvent (25% v/v ethanol, 8% v/v acetic acid). The molecular weight of the collagen fractions was established using molecular weight standards from AMRESCO (Protein MW Marker, Wide Range): myosin (212 kDa), β-galactosidase (116 kDa), phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31.0 kDa), and soybean trypsin inhibitor (21.5 kDa).

Protein extracts were separated by SDS-PAGE in 8% slab gels (12 mA, 30 min; 30 mA, 90 min) in a MiniProtean II Cell system (BioRad). 20 µg protein extract was loaded per well including 1 µl Benzonase Nuclease HC (Novagen, Madison, WI). Blotting onto 0.45 µm nitrocellulose membranes (BioRad) was performed at 100 V for 1 h in a Mini Trans-Blot Cell wet transfer system (BioRad). Membrane blocking was carried out with 5% skimmed milk in PBS-0.1% Tween 20 (Sigma) for 1 h and washed three times with PBS-1% Tween 20 for 15, 5 and 5 min respectively. Next, membranes were incubated with 1∶500 of rabbit anti-LACK polyclonal serum for 2 h [18] (link) or 1∶10,000 of monoclonal mouse anti-L. mexicana glycosomal GAPDH antibody kindly provided by Paul Michels (University of Edinburg) [19] (link), washed again and incubated with 1∶2,000 HRP-conjugated goat anti-rabbit IgG (DAKO, Ely, UK) for 90 min. Once the wash steps were repeated, the immunoblots were developed using the ECL detection system (GE Healthcare, Pittsburg, PA) according to the manufacturer's instructions.

Digestion products were analyzed by SDS PAGE under reducing conditions. The analysis was carried using a BioRad MiniProtean II cell system and BioRad TGX gradient precast gels (any kDa or 4–20%, BioRad Laboratories, Hercules, CA, USA) or 16% acrylamide hand-cast gels.