Novex precast 4 20 tris glycine polyacrylamide gels

Myofibrillar protein fractions were isolated from 2 g of minced meat according to the procedure of Pietrzak et al. (1997) (link). A biuret assay was performed to determine protein concentrations. Samples were then diluted to a protein concentration of 2 mg/ml in sample buffer (8 mol urea, 2 mol thiourea, 3% SDS (wt/vol), 75 mmol DTT, 25 mmol Tris-HCl (pH 6.8), 0.004% bromophenol blue) and denatured for 3 min in boiling water. Denatured protein samples (15 µg protein/lane) and a broad range molecular weight standard (5–250 kDa, Thermo Scientific PageRuler Broad Range Unstained Protein Ladders, Waltham, MA, United States) were loaded onto Novex precast 4%–20% tris-glycine polyacrylamide gels (Life Technologies Corp., Carlsbad, CA, United States) and ran at 4°C at a constant voltage. Gradient gels were utilized to allow for a broader range of proteins to be analyzed, and equal protein loads ensured that differences were because of actual variations in the protein profiles. Gels were then stained (Coomassie brilliant blue R-250) and destained. The densities of 13 myofibrillar protein bands were quantified using Alpha View software (v 3.4, ProteinSimple Inc., Santa Clara, CA, United States). The relative abundance of each individual protein band was expressed as a percentage of the total protein abundance of all bands within the lane.