We improved the separation of the basic proteins from the soybean cotyledon, Glycine max L. Merr. by searching N-terminal sequences data in proteins isolated by two-dimensional electrophoresis (2-DE). After removed Hexane, proteins were extracted from cotyledon with a urea/Triton/2-mercaptoetanol solution. Using this method, the highly reproducible isoelectric focusing (IEF) can formed with polyacrylamide gels with pH 4.0-9.8. The IEF tube gels were used as the first dimension, and proteins were visualized by second-dimensional gel electrophoresis, and identify a number of soybean cotyledon proteins using mass spectrometry in the proteome analysis. These instruments of 2-DE and IEF tube gels were used 27 cm and investigate under various conditions. The total number of spots and features was obtained by PDQuest software (Bio-Rad). In this experiments performed, the IEF tube gels and instruments afforded good reproducibility in the number of PDQuest-detected spots from gel to gel while IPG offered better reproducibility in the total number of manually detected spots from gel to gel. In conclusion, we have separated of the basic 13 proteins in soybean. The glycinin subunit separations are also considered to play important roles in soybean breeding and biochemical characterization. The improved technique will be useful to dissect the genetic control of glycinin expression in soybean.