Improved sodium dodecyl sulfate-polyacrylamide gel electrophoresis method for quantifying the 11S/7S ratio in soybean...

Accurate quantification of soybean storage protein subunits glycinin (11S) and β-conglycinin (7S), and their ratio (11S/7S), is essential for applications in plant breeding, food processing, and allergen research. Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is widely used due to its accessibility and cost-effectiveness, its quantitative reliability is often limited by variability in protein extraction and band resolution. This study presents an improved SDS-PAGE protocol optimized for quantifying the 11S/7S ratio with improved accuracy, reproducibility, and cost-effectiveness.

Two established extraction protocols were evaluated across three soybean cultivars, and a new protocol was developed by integrating their strengths. The new protocol employs a Tris-HCl-based buffer containing sucrose, ethylenediaminetetraacetic acid, β-mercaptoethanol, and phenylmethylsulfonyl fluoride, which improves protein solubility and minimizes degradation. Compared to two prior protocols, the new method achieved clearer band resolution, lower error variance, and higher F-values in analysis of variance. Standard curves derived from bovine serum albumin confirmed strong linearity between band intensity and protein quantity (r² = 0.993, 0.985), supporting reliable quantitative analysis. Reproducibility tests demonstrated consistent 11S/7S estimation across independent runs. In an application evaluation across 14 breeding lines, the protocol effectively discriminated genotypic variation, with 11S/7S ratio ranging from 1.29 to 2.20 and separation into seven statistically distinct groups, which further validated the protocol's stability and discriminatory power.

This optimized SDS-PAGE method provides a reliable, scalable tool for phenotyping 11S/7S ratios in diverse soybean germplasm, offering a practical balance between resolution, affordability, and throughput for both research and applied use. © 2026 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.