Identification of Soybean Lipoxygenase Isozyme by Native-PAGE

There are some problems existed in the detection of soybean lipoxygenase(Lox)isozymes such as complex procedures,poor resolution,higher cost and highly toxic reagents on the human,exploring an improved methods to identify Lox isozyme of soybean is necessary.In this study,Lox deletion mutants(Wuxing No.1,Wuxing No.3 and Wuxing No.4)and non-deletion type(Zhonghuang No.13)were used to survey the optimal experimental condition for Lox ioszymes by changing the separation gel concentration,electrophoresis voltage,sample concentration,time and staining method with Native-PAGE.From the results we found that the four Lox isoenzyme of soybean(Lox1,Lox2,Lox3a and Lox3b)could be separated clearly when the concentration of stacking gel and separating gel were 5% and 13%,repectively;electrophoresis voltage in stacking gel and separation gel were 90 V and 190 V,respectively;sample concentration was 40%,and the electrophoresis time was 7 hours,dying with two different enzyme staining of pH8.0 and pH6.5 combined with heat Coomassie blue staining.In short,it is a simple,safe,economical and effective method that can be used for the identification and screening of lipoxygenase deletion mutants of soybean germplasm.