Protein assay kit 2 500 0002

Myofibril proteins were isolated in accordance with the method of Morzel et al. [26 (link)] with slight modification as outlined by Nakyinsige et al. [27 (link)]. Approximately 2.5 g of pulverized muscle tissue was homogenized for 30 s on ice in 25 ml of extraction buffer containing 25 mM KCl, 150 mM NaCl, 4 mM EDTA, and 3 mM MgCl₂ at pH 6.5 to which protease inhibitor (CALBIOCHEM®, Cat # 55140, EMD Bioscience, Inc. Germany) was added. After filtration, the homogenate was incubated at 4°C with stirring. This was followed by centrifugation at 2000 g for 15 min at 4°C using an Avanti^® J-26XPI centrifuge (BECKMAN COULTER®, USA). The pellets were washed twice with 25 ml of a 50 mM KCl solution at a pH of 6.4 and once with 25 ml of 20 mM phosphate buffer of pH 6. The pellets were finally re-suspended in the same phosphate buffer and the protein concentration of the samples was estimated in accordance to the Bradford method [28 (link)] with the aid of Protein Assay Kit II 500–0002 (Bio-Rad, USA) following the micro plate protocol for colorimetric procedure.

Myofibrillar proteins were isolated from pulverized muscles (GM, n = 20; IS, n = 20 at each ageing period) as described by Morzel et al. [31 (link)]. Pulverized muscle (2.5 g) was homogenized for 30 s on ice with 25 mL extraction buffer (25 mM KCl, 150 mM NaCl, 4 mM EDTA, 3 mM MgCl₂, at pH 6.5) to which protease inhibitor (CALBIOCHEM®, Cat # 55140, EMD Bioscience, Inc. Germany) was added. The homogenate was filtered through 1.0 mm polyethylene strainer to eliminate any remaining collagen. After filtration, the homogenate was incubated at 4 °C with continuous shaking. This was followed by centrifugation at 2000 g for 15 min at 4 °C. The pellet was washed twice with 25 mL of a 50 mM KCl solution at pH 6.4 and once with 25 mL of 20 mM phosphate buffer at pH 6. The pellet was homogenized (T18 digital ULTRA-TURRAX® - IKA, Germany) in 5 mL of 20 mM phosphate buffer for 1 min on ice. The homogenate was centrifuged (Avanti® J-26XPI, BECKMAN COULTER®, USA) at 15,000 g for 20 min at 4 °C. The total protein concentration of an aliquot of the clear supernatant was determined by the method of Bradford [32 (link)] using Protein Assay Kit II 500–0002 (Bio-Rad, USA). The protein standards were prepared with Bovine serum albumen [33 ].