副溶血弧菌总蛋白双向电泳体系的建立
首发时间:2012-11-01
摘要:以副溶血弧菌为研究对象,从样品制备、裂解液配方、IPG胶条长度、IPG胶条pH范围、水化上样方式、等电聚焦程序、上样量和二向胶浓度等因素的对比试验,利用超声破碎与TCA沉淀相结合的样品制备方法,使用17cm pH4~7的IPG胶条,采取主动水化上样,上样量为100μg,上样体积为300μL,适当延长除盐时间(2.5h)和等电聚焦时间(80000 vhr),采用12.5%浓度的二向分离胶,建立并优化了副溶血弧菌总蛋白质双向电泳技术体系,为后续差异蛋白质组学研究奠定基础。
关键词: 食品安全 副溶血弧菌 蛋白质 双向电泳(2-DE)
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Establishment of two-dimensional gel electrophoresis (2-DE) system for protemics analysis of the total protein in Vibrio parahaemolyticus
Abstract:The total proteins extracted from Vibrio parahaemolyticus were separated by carrierampholytes pH gradients based immobilized isoelectric focusing gel electrophoresis for first and the vertical SDS-polyacrylamide gel electrophoresis for second to the line of bidirectional gel electrophoresis two-dimensional gel electrophoresis (2-DE). The gels were dyed and scanned to gain gel image, then PD-Quest software was used for gel image analysis. The 2-DE related techniques were constructed and optimized by comparative tests on some important factors including different extraction methods, lysis buffer components, length of IPG strips, pH range of IPG strips, isoelectric focusing programs, sample volume, and so on. The results showed that the resolution and reproducibility of 2-DE profiles was significantly improved by adding Tris and TBP in lysis buffer, the combination of sonication and trichloroacetic acid/acetone (TCA) precipitation for sample preparation, active rehydration of 17cm (pH 4-7) IPG gel strips, loading the sample 100μg with sample volume of 300μl, prolonging the time of desalting (2.5 h) and isoelectric focusing time (80000 vhr), preparing 12.5% SDS-PAGE gel, and dying the gels by the silver stained. This work provided a technical basis for the further study on differential proteomics of V. parahaemolyticus.
Keywords: Food safety Vibrio parahaemolyticus total protein two-dimensional gel electrophoresis (2-DE)
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