Establishment of two-dimensional electrophoresis (2-DE) technique in muscle proteome of Fenneropenaeus chinensis.

Abstract

In order to use two-dimensional electrophoresis(2-DE)analysis in the muscle proteins of Chinese shrimp(Fenneropenaeus chinensis),its muscle proteins were separated using immobilized pH gradient 2-DE after dissolving.By optimizing different extraction methods,pH of IPG gel strips and its length,isoelectric focusing programs,and loading amount,etc,the proteins were successfully extracted from Chinese shrimp muscle and were separated by 2-DE.After silver staining or Coomassie brilliant blue staining,PDQuest image analysis software was applied to analyze the 2-DE images.The results showed that most of shrimp muscle protein isoelectric points were between 4 and 7.The 2-DE related techniques was constructed and optimized in muscle proteome of Chinese shrimp by comparative tests on different extraction methods,IPG gel strips,isoelectric focusing programs,salt bridge,and sample volume,etc.The results showed that the resolution and reproducibility of 2-DE profiles were significantly improved by adding thiourea,CHAPS and DTT in lysis buffer,active rehydrating of 17 cm(pH 4-7)IPG gel strips,loading the sample 120 μg,prolonging the time of desalting,increasing the voltage and power of isoelectric focusing,employing the salt bridge,preparing 12.5% SDS-PAGE gel,and dying the gels by the silver staining.It shows that the repeatability and stability are good enough.