Abstract: [Objective] To explore the extraction methods of protein samples for SDS-PAGE and two-dimensional electrophoresis (2-DE) of the stem, vegetable, and vegetable seeds, and lay the foundation for the study of seed protein proteomics of the two species of Lepidium plants.[Method]The efficiency of four protein extraction methods were compared and analyzed through protein purity detection and SDS-PAGE.[Result]Of all the four methods, TCA/acetone extraction method generated higher yield as well as highest resolution and clear estelectrophoresis bands in SDS-PAGE, lysis extraction method got the highest protein yield whereas with highest impurities, Tris-HCl method and phenol method could obtain the highest purity but lowest protein yield, meanwhile protein extracted by lysis method and phenol method had the highest level of high-abundance proteins which could cause serious interference in further SDS-PAGE and 2-DE analysis.We got 6 differential expressed bands after compared between two species of Lepidium plants by SDS-PAGE using seed total protein extracted by TCA/acetone extraction method and phenol method.[Conclusion]TCA/acetone seed total protein extraction method is an optimal sample preparation protocol for SDS-PAGE and 2-DE.Besides, the differential bands of SDS-PAGE electrophoresis of two kinds of Lepidium seed protein could provide reference for screening two seeds.