Plant Protein Extraction and Characterization

The plant tissue (1g) was extracted using freshly prepared protein extraction buffer as previously described by Muneer et al., 2016 (link). The extraction buffer constituted (pH 7.5) 40 mM (w/v) Tris-HCl (pH 7.5), 2 mM (w/v) EDTA, 0.07% (w/v) β-mercaptoethanol, 2% (w/v) PVP and 1% (v/v) Triton X-100. The extract was centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was mixed with protein-dye and 20 µg proteins were loaded on 12.5% polyacrylamide gel on PROTEAN II (Bio-Rad, Hercules, CA, USA). The protein concentration was determined by the Bradford method using BSA (bovine serum albumin) as a standard curve. Following electrophoresis, the gel was stained with commercially available Coomassie brilliant blue stain (CBBS) according to manufacturer’s instruction (Bio-Rad).