Total protein extracts from lyophilized meat samples were obtained according to Franco et al. [35 (link)]. Extraction and protein purification were performed with the Clean-Up kit (GE Healthcare, Uppsala) from crude cell lysates obtained by ultrasonic disruption using a Branson digital sonifier (model S-250, Branson Ultrasonics, Danbury). Protein concentration in samples was assessed with an improved Bradford method using the CB-X protein assay kit (G-Biosciences, St. Louis) following the instructions of the manufacturer to remove interfering agents and use with a microplate reader. The bovine serum albumin (BSA) protein standard was used to determine protein concentration from calibration curves.
Proteins from lyophilized meat samples were separated by 2-DE as previously described by Franco et al. [35 (link)]. Briefly, 350 μg of each biological replicate were loaded onto an immobilized pH gradient (IPG) strip (24-cm long, pH 4–7 linear gradient, ReadyStrip IPG strips, Bio-Rad Laboratories, Hercules). First-dimension isoelectric focusing (IEF) of proteins in strip gel was performed using a PROTEAN IEF cell system (Bio-Rad Laboratories). The second dimension was run on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using an Ettan DALTsix large vertical electrophoresis system (GE Healthcare).
Proteins from lyophilized meat samples were separated by 2-DE as previously described by Franco et al. [35 (link)]. Briefly, 350 μg of each biological replicate were loaded onto an immobilized pH gradient (IPG) strip (24-cm long, pH 4–7 linear gradient, ReadyStrip IPG strips, Bio-Rad Laboratories, Hercules). First-dimension isoelectric focusing (IEF) of proteins in strip gel was performed using a PROTEAN IEF cell system (Bio-Rad Laboratories). The second dimension was run on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using an Ettan DALTsix large vertical electrophoresis system (GE Healthcare).