Gel electrophoresis
The native protein in ground defatted silflower seed showed a thin, defined band that resolved at just above 242 kDa, a dark colored area between 300 and 720 kDa, and a faint band at around 800 kDa (Fig. 3, lane 2). The high molecular weight of the dominant polypeptide (300-720 kDa) may indicate a cross-linked structure. The albumin fraction (lane 3) showed only one band near the 146 kDa marker. The globulin fraction’s (lane 4) two obvious major bands resolved at 66 and 146 kDa, but there was one faint band near 50 kDa and four faint bands between 246 and 720 kDa. Silflower albumin has higher molecular weight than sunflower albumin (10-18 kDa), but the globulin is smaller than sunflower globulin (helianthinin, 300-350 kDa) (Gonzalez-Perez and Vereijken, 2007). There was no defined band detected for the prolamin fraction, although a “shadow” is noticeable near the 720 kDa marker (lane 5). The glutelin fraction (lane 6) polypeptide had the lowest molecular weight, as indicated by the dark area that resolved between 10 and 66 kDa. For purification of silflower protein extracts, membrane with MWCO <50 kDA is appropriate for saline-extracted proteins (albumins and globulins) and MWCO <10 kDA for alkali-extracted proteins.
In reducing gel electrophoresis, there were 13 polypeptide bands detected in defatted silflower meal (Fig. 4, lane 2). Their molecular weight range was 13-140 kDa, with the six darkest and/or widest bands resolving around 15-20, 37, and 50-60 kDa. The water-soluble fraction showed one dark band near 15 kDa and eight faint and narrow bands that resolved between 18 and 50 kDa. The NaCl-soluble group (lane 4) showed eight polypeptide bands from 5-75 kDa, with the five most dense bands appearing at the lower molecular weight markers (5, 10, 20, 30, and 40 kDa). The ethanol-soluble (lane 5) and NaOH-soluble (lane 6) fractions showed the least number of polypeptide bands with three each and both sets resolving between 15-30 kDa. In the spent solids (lane 7), only two faint bands near the 20 kDa mark were observed, indicating that sequential solvent extraction removed much of the protein in the defatted silflower meal.