DefinePK

Abstract

Background: Seed proteins are essential biomolecules contributing to plant physiology and offering significant nutritional and therapeutic benefits to humans. They serve as a vital source of amino acids and display bioactive properties, including antioxidant and immunomodulatory effects. Accurate profiling of seed proteins is critical for nutritional assessment, quality control, and botanical authentication. Among various analytical methods, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is widely used due to its reliability, affordability, and resolution capacity in differentiating protein patterns across species.
Objective: This study aimed to analyze and compare the protein expression profiles of Nigella sativa (kalonji), Allium cepa (onion), and Linum usitatissimum (flaxseed) using SDS-PAGE and densitometry, to identify interspecies variation and establish protein abundance for seed authentication and nutritional insights.
Methods: Certified seeds were defatted with hexane, homogenized in 0.1 M Tris-HCl buffer (pH 8.0) with 1 mM EDTA, and centrifuged at 10,000 × g for 15 minutes. Protein quantification was performed using the Bradford assay. SDS-PAGE was carried out with 20 µg of protein per lane, followed by Coomassie Brilliant Blue R-250 staining and destaining. Densitometry was conducted using ImageJ software to calculate relative protein abundance units (RPAU), with normalization based on flaxseed.
Results: Flaxseed displayed the highest total intensity (F1: 63%, F2: 60%) and RPAUs of 100 and 95, with estimated protein concentrations of 10.0 and 9.5 mg/mL. Kalonji samples showed intensities of 54% and 52%, RPAUs of 86 and 83, and concentrations of 8.6 and 8.3 mg/mL. Onion samples exhibited lower values at 52% and 51%, with RPAUs of 83 and 81, corresponding to 8.3 and 8.1 mg/mL. Inter-duplicate variance remained under 5%, indicating strong reproducibility.
Conclusion: SDS-PAGE combined with densitometry proved effective in distinguishing protein profiles across seeds. The findings support its continued use in biochemical fingerprinting, food authenticity, and nutritional evaluation of botanicals.