The soluble soybean protein in SBM,

The soluble soybean protein in SBM, FSBM and SPC were extracted by the method of Faurobert (1997). One hundred miligram sample mixed thoroughly with 400 μl of protein extraction solution (50 mM Na2CO3, 100 mM NaCl, 0.05% Triton X-100, 0.05% Tween-20, 1 mM PMSF, 1% βmecaptoethanol) for 10 min. The slurry was centrifuged at 7,000×g for 30 min. The supernatant was collected and the protein concentration was measured by the method of Bradford (1976) using BSA as the standard. Soluble protein was fractionated by a Tricine-SDSPAGE system according to the method described by Schagger (2006). The electrophoresis system was based on 10% polyacrylamide separating gels containing 0.1% SDS in Tris- tricine buffer. About 30 μg of extracted soluble protein was loading for each well and the sample was separated at 50-60 mV for 5 h. The protein markers (Invitrogen, USA) with the following molecular weights
Chen et al. (2010) Asian-Aust. J. Anim. Sci. 23(5):598-606
600
include Myosin (210.0 kDa), Bovine serum albumin (78.0 kDa), Glutamic dehydrogenase (55.0 kDa), Alcohol dehydrogenase (45.0 kDa), Carbonic anhydrase (34.0 kDa), Myoglobin (23.0 kDa), Lysozyme (16.0 kDa), Aprotinin (7.0 kDa) and Insulin B chain (4.0 kDa). After electrophoresis, the gel was stained for 30 min using Coomassie Brilliant Blue R-250 and de-stained with acetic acid. Images of the gels were captured with densitometers (Astra 2200, UMAX®) and analysis with software (Gel pro 4.5v, Media Cybernetics®) to calculate relative percentage of each protein band in test samples.