食品科学2017，Vol.38Issue(6)：27-35,9.DOI:10.7506/spkx1002-6630-201706005
基于荧光标记的大黄鱼氧化肌肉蛋白质双向电泳技术的建立
Establishment of Two-Dimensional Gel Electrophoresis Based on Fluorescence Labeling for Oxidized Muscle Proteins of Large Yellow Croaker (Pseudosciaena crocea)
摘要
Abstract
The purpose of this study was to optimize the processing parameter for fluorescence labeling of oxidized muscle proteins from large yellow croaker (Pseudosciaena crocea) with luorescein-5-thiosemicarbazide (FTSC),and further to establish a two-dimensional gel electrophoresis (2-DE) system for oxidized muscle proteins.The results showed that the optimized electrophoresis process was as follow.The protein sample was prepared by liquid nitrogen milling and using a lysis buffer containing 8 mol/L urea,2 mol/L thiourea,4％ CHAPS,65 mmol/L DTT and 0.2％ carrier ampholyte,and then the oxidized protein was labeled with FTSC (20 mmol/L in DMSO) for 3 h at 40 ℃ in the dark,and washed five times with ethanol/ethyl acetate (1∶1).The sample was loaded onto immobilized pH gradient (IPG) gel strip (pH 5-8),and separated by isoelectric focusing (active rehydration for 14 h at 50 V;desalting for 2 b at 500 V,1.5 h at 1 000 V and 1 h at 4 000 V;voltage for 0.5 h at 6 000 V at first and then for 1 h at 10 000 V;focusing for 80 000 vhr at 10 000 V;finally balancing for 10 h at 500 V).After isoelectric focusing,the IPG strip was transferred and the proteins were separated by 12％ SDS-PAGE.Finally,2-DE maps for the oxidized and whole muscle proteins with high resolution and even distribution were obtained by direct scanning using a fluorescence image scanner and scanning after silver staining.This study would provide a foundation for the separation and identification of oxidized muscle proteins,and further for the clarification of protein oxidation mechanism by two-dimensional gel electrophoresis and proteomics technology.关键词
双向电泳/大黄鱼/氧化肌肉蛋白质/荧光素-5-氨基硫脲/荧光标记Key words
two-dimensional electrophoresis/large yellow croaker (Pseudosciaena crocea)/oxidized muscle proteins/fluorescein-5-thiosemicarbazide (FTSC)/fluorescence labeling分类
轻工纺织引用本文复制引用
李学鹏,周明言,渠宏雁,王金厢,朱文慧,徐永霞,仪淑敏,林洪,励建荣..基于荧光标记的大黄鱼氧化肌肉蛋白质双向电泳技术的建立[J].食品科学,2017,38(6):27-35,9.基金项目
国家自然科学基金青年科学基金项目(31301569) （31301569）
国家自然科学基金面上项目(31571868) （31571868）
中国博士后科学基金面上资助项目(2015M582143) （2015M582143）
“十二五”国家科技支撑计划项目(2015BAD17B03) （2015BAD17B03）
辽宁省教育厅重点实验室基础研究项目(LZ2014047) （LZ2014047）
