To optimize the conditions for the establishment of two-dimensional gel electrophoresis (2-DE) of soybean protein isolate (SPI), we investigated Ampholine mixture, anodic and cathodic electrolytes, loading amount of sample, acrylamide concentration, pH gradient and gel staining method in two-dimensional gel electrophoresis to optimize the protein imaging conditions in two-dimensional gel. The results of mixed-level design experiments showed that Ampholine, loading amount and gel staining method had significant effect (P