Quantifying Wheat Storage Proteins

HPLC was used to determine relative quantities of gliadins and glutenins in a sample. To obtain a standard curve using HPLC, increasing concentrations of bovine serum albumin (BSA; 0, 10, 25, 50, 75, 100, 150, and 300 mg/mL) were loaded onto the C8 reversed-phase analytical column (Zorbax 300SB-C8, Agilent Technologies) and resolved following Mejías et al. (38 (link)). Two peaks were observed, respectively, at 16.78 and 17.98 min retention times. Peaks were integrated, and the obtained peak areas in milli-absorbance units (mAU) were regressed against the BSA concentrations loaded onto the HPLC column to obtain a standard curve. The gliadin and glutenin extracts of the control and DME TILLING mutants were analyzed analogously as mentioned above for BSA, and the peak areas were calculated. The mAU values were plotted on the standard curve to determine relative protein concentrations, and the values obtained for each peak were summed to calculate a cumulative concentration for each of the glutenin and gliadin fractions from control and mutant lines.