Chemidocxrs gel imaging analysis system

The protein components of roasted pumpkin seeds were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of electrophoresis gel was 12.5% and that of concentrated gel was 4%. The pumpkin seed protein dissolved in the pH 7.0 phosphate buffer solution whose content was 5 mg/mL determined by microplate (Molecular Devices, LLC., California, USA)was mixed with the sample buffer (5 ×, 250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β- mercaptoethanol) at the volume ratio of 4:1. The mixed sample was soaked in boiling water for 10 min and was loaded (10 μL). The gels run at the voltage of 120 V with Tris-Glycine buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). After electrophoresis, the SDS-PAGE gel was stained with Coomassie brilliant blue G-250 for 2 h, and compare with a protein maker by ChemiDocXRS+ gel imaging analysis system (Bio-Rad Laboratories, Inc., California, USA) after decolorization. The protein marker purchased from Epizyme Biotechnology Co., Ltd (Shanghai, China) consists of 11 protein bands ranging from 10 to 250 kDa.

Briefly, extract cardiac tissue protein, and the protein concentrations were assessed using the Bradford assay (Roche). Proteins were separated using SDS-PAGE electrophoresis and transferred to PVDF membranes. Membranes were blocked with nonfat milk and probed with primary polyclonal antibodies. Membranes were incubated with HRP-conjugated anti-rat IgG. Antibody-bound protein bands on the immunoblot were visualized using a Chemi DOC XRS+ Gel imaging analysis system (BIO-RAD, USA).