2.4.3. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

To confirm the formation of covalent bonding, the electrophoretic profiles of conjugate samples were examined by the method of Laemmli (1970) with minor modifications. Rice protein hydrolysates and their conjugates were dispersed in the loading buffer containing 0.5 M Tris-HCl (pH 6.8), 1% sodium dodecyl sulfate (SDS), 5% (v/v) β-mercaptoethanol, 1% (w/v) bromophenol blue, and 10% (v/v) glycerol. The mixtures were incubated in boiling water for 5 min, and after being cooled down to room temperature, it was centrifuged at 10,000 × g for 10 min. The conjugate samples (containing 30 μg of protein), along with standard protein marker (5–245 kDa), were subjected to SDS-PAGE run at a constant voltage of 80 V. After electrophoresis, the gels have stained with a solution containing 0.5% (w/v) Coomassie Brilliant Blue R-250, 60% (v/v) methanol and 5% (v/v) acetic acid for 3 h and were then detained in the solution containing 60% (v/v) methanol and 5% (v/v) acetic acid for 4 h.