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.