2.4 Recombinant Protein extraction
To find out the accumulation of recombinant fusion proteins, 50 mg of seed powder was used to mix with 500 µl of N -hexane followed by centrifugation for 5 min at 5000×g, and supernatant was discarded. The process is repeated 3 times and fine seed powder was dried in vacuum. Double dH2O was added to powder with 1/10 (w/v) seed weight/water ratio and were incubated for 1 hour with agitation, followed by centrifugation of 13000 rpm at 4°C. The samples was homogenized with 3 volume of protein extraction buffer (3% (w/v) CTAB, 1,4 M NaCl, 0,2 % (v/v) β-Mercaptoethanol, 20 mM EDTA, pH 8.0, 100 mM Tris-HCl, pH 8.0, 1 % (w/v) PVP40). Centrifuge for 15sec and the 100ul of supernatant was added to new 1.5 Eppendorf tube. Centrifuge again for 40 min at 13,000 rpm at 4°C. The supernatant will be discarded, and the pellet will be re-suspended in 5X loading buffer for further analysis. The total soluble protein (TSP) concentration of the extracts was determined by Bradford assay.