大豆蛋白提取.docx

3.8. Protein Extraction
Total proteins from different samples (0.3 g) were extracted in 1 mL cold TPBS buffer (150 mM NaCl, 8 mM Na2HPO4·12H2O, 2 mM KH2PO4·H2O, 4 mM KCl, 0.05% Tween-20, and 2% SDS, pH 7.4). After shaking for 30 minutes at room temperature, the mixture was centrifuged twice at 20,000 g in a rotor (model Avanti J-25, Beckman) for 15 min each. Total protein concentration was determined by BCA (Bicinchoninic acid) assay (Multisciences Biotechnology Co. Ltd., Hangzhou, China), using bovine serum albumin (BSA) as a standard.
3.9. Western Blotting Analysis
The predicted protein product of CP4-EPSPS comprises 527 amino acids with molecular weight of 55.5 kD, which includes a 72 amino acid chloroplastic transit peptide proven previously [13], indicating a mature protein of 47.6 kD following the cleavage site of the transit peptide. In our experiment, one peptide sequence containing the specific amino acids corresponding to the mature CP4-EPSPS sequence positions (S19→R33) for the antigen were obtained by chemical synthesis. The peptide was hydrophilic, surface-oriented, and flexible. Synthetic peptides were purified by HPLC and coupled to keyhole limpet haemacyanin (KLH). The CP4-EPSPS-KLHs were collected and used for producing the rabbit polyclonal antibody (SC-16). For the western blot analysis, protein (30 μg) or protein extracted from an equal weight of different
samples was subjected to SDS-PAGE using a 12.5% acrylamide resolving gel (Mini Protean II System, Bio-Rad, Hertz, UK). Separated proteins were then transferred to polyvinylidene difluoride (PVDF) membranes, and non-specific binding of antibodies was blocked with 5% non-fat dried milk in phosphate-buffered saline (PBS, pH 7.4) overnight at 4 °C. Membranes were then incubated overnight at 4 °C, with primary antibodies diluted 1:3000 in PBS buffer plus 1% non-fat dried milk. Immune complexes were detected using horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G. The color was developed with a solution containing 3,3'-diaminobenzidine tetrahydrochloride (DAB) as the HRP substrate. Meanwhile, identical SDS-PAGE gel was stained by Coomassie Brilliant Blue.