高白鲑肌肉蛋白质组双向电泳技术体系的优化
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摘要
为建立高白鲑肌肉蛋白质双向电泳分析方法,通过固相p H梯度胶条等电聚焦和SDS-PAGE垂直电泳对蛋白质进行分离,分别考察了蛋白质样品制备方法、上样量、IPG胶条p H值、等电聚焦程序、助凝剂剂量、SDS-PAGE凝胶浓度及染色方法等影响的因素。结果显示,采用丙酮沉淀法制备蛋白质样品,上样量90μg,IPG胶条pH值5～8,一向胶聚焦时间400 V 16 h+1 000 V 2 h,平衡时间15 min,0.05 m L过硫酸铵及2μL TEMED,10%(体积分数)二向分离胶和硝酸银染色时,获得蛋白质分离效果好,分辨率高及横纹少的高白鲑肌肉蛋白质二维电泳图谱,为高白鲑肌肉蛋白质组学的进一步研究奠定了基础。
In order to establish a two-dimensional electrophoresis(2-DE) system for proteomic analysis of Coregonus peled muscle,its muscle proteins were separated using immobilized pH gradient 2-DE.Explored the influence factors such as preparation methods,loading amount,p H of immobilized p H gradients(IPG) gel strips,isoelectric focusing programs,the choice of coagulant aid,the concentration of SDS-PAGE gel and staining methods.The results show that the optimum method for protein samples preparation of Coregonus peled muscle was acetone precipitation.For 2-DE assay,the loading amount was 90 μg,using pH 5-8 IPG gel strips for isoelectric focusing(IEF),time of electrophoresis was400 V 16 h + 1 000 V 2 h and balanced with 15 min,concentrations of assistant reagents were 0.05 mL ammonium persulfate and 2 μL TEMED,using 10% gel for the second SDS-PAGE and dying the gels by the silver staining.In this condition,we finally got satisfactory 2-DE patterns with good protein separation degree,high repetition and less band,which stands as a valuable resource for further proteomics research of Coregonus peled.
In order to establish a two-dimensional electrophoresis(2-DE) system for proteomic analysis of Coregonus peled muscle,its muscle proteins were separated using immobilized pH gradient 2-DE.Explored the influence factors such as preparation methods,loading amount,p H of immobilized p H gradients(IPG) gel strips,isoelectric focusing programs,the choice of coagulant aid,the concentration of SDS-PAGE gel and staining methods.The results show that the optimum method for protein samples preparation of Coregonus peled muscle was acetone precipitation.For 2-DE assay,the loading amount was 90 μg,using pH 5-8 IPG gel strips for isoelectric focusing(IEF),time of electrophoresis was400 V 16 h + 1 000 V 2 h and balanced with 15 min,concentrations of assistant reagents were 0.05 mL ammonium persulfate and 2 μL TEMED,using 10% gel for the second SDS-PAGE and dying the gels by the silver staining.In this condition,we finally got satisfactory 2-DE patterns with good protein separation degree,high repetition and less band,which stands as a valuable resource for further proteomics research of Coregonus peled.
引文
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[12]GRG A,BOGUTH G,OBERMAIER C,et al Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension(IPG-DALT):the state of the art and the controversy of vertical versus horizontal systems[J].Electrophoresis,1995,16(1):1079-1086.
[13]ONG S E,PANDEY A.An evaluation of the use of two-dimensional gel electrophoresis in proteomics[J].Biomolecular Engineering,2001,18(5):195-197.
[14]吴海庆,李明云,叶帅东,等.大弹涂鱼肝脏蛋白质双向电泳技术的建立及优化[J].生命科学仪器,2009,7(8):29-32.
[15]高艳利,杨思文,樊凯奇,等.SDS-PAGE电泳技术分析蛋白质的研究[J].辽宁化工,2007,36(7):460-463.
[16]PINK M,VERMA N,RETTENMEIER A W,et al CBB staining protcol with higher sensitivity and mass spectrometric compatibility[J].Electrophoresis,2010,31(4):593-598.
[17]马增春,高月,吕俊萍,等.双向凝胶电泳中三种蛋白质检测方法的比较[J].中国生物化学与分子生物学报,2004,20(4):551-556.
[2]李学鹏.中国对虾冷藏过程中品质评价及新鲜度指示蛋白研究[D].杭州:浙江工商大学,2012.
[3]RODRIGUES P M,SILVA T S,DIAS J,et al Proteomics in aquaculture:Applications and trends[J].Journal of Proteomics,2012,75(14):4325-4345.
[4]徐永杰.猪肌肉组织双向电泳分离条件的建立及常见问题分析[J].农业生物技术学报,2012,20(1):105-112.
[5]梁光明.凡纳滨对虾肌肉蛋白质双向电泳分离技术的建立及优化[J].中国食品学报,2015,15(10):240-245.
[6]王任,王彦波.大黄鱼肌肉组织双向凝胶电泳方法的建立[J].中国食品学报,2012,12(11):155-159.
[7]丁勤学.不同上样方式对蛋白质组双向电泳图谱质量的影响比较[J].中国生物化学与分子生物学报,2006,22(3):239-242.
[8]郭焱,马燕武,蔡林刚,等.赛里木湖高白鲑和凹目白鲑肌肉、卵的营养分析评价[J].水产学杂志,2004,17(1):62-67.
[9]宋广磊,戴志远.鱼肉蛋白质冷冻变性研究进展[J]食品科技,2006,31(3):17-20.
[10]王瑞,傅玲琳,王彦波.副溶血弧菌总蛋白双向电泳体系的建立[J].中国食品学报,2013,13(12):146-155.
[11]储兆东,卢国华,谭英,等.三氯乙酸/丙酮蛋白质提取法在白内障晶状体蛋白提取中的应用[J].国际眼科杂志,2013,13(12):2377-2380.
[12]GRG A,BOGUTH G,OBERMAIER C,et al Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension(IPG-DALT):the state of the art and the controversy of vertical versus horizontal systems[J].Electrophoresis,1995,16(1):1079-1086.
[13]ONG S E,PANDEY A.An evaluation of the use of two-dimensional gel electrophoresis in proteomics[J].Biomolecular Engineering,2001,18(5):195-197.
[14]吴海庆,李明云,叶帅东,等.大弹涂鱼肝脏蛋白质双向电泳技术的建立及优化[J].生命科学仪器,2009,7(8):29-32.
[15]高艳利,杨思文,樊凯奇,等.SDS-PAGE电泳技术分析蛋白质的研究[J].辽宁化工,2007,36(7):460-463.
[16]PINK M,VERMA N,RETTENMEIER A W,et al CBB staining protcol with higher sensitivity and mass spectrometric compatibility[J].Electrophoresis,2010,31(4):593-598.
[17]马增春,高月,吕俊萍,等.双向凝胶电泳中三种蛋白质检测方法的比较[J].中国生物化学与分子生物学报,2004,20(4):551-556.