近红外成像用于双向电泳前的蛋白质快速定量  

Rapid Protein Quantitation in Pre Two-dimensional Electrophoresis by Near-Infrared Imaging

作　　者：刘云[1] 曹嵩 李科[3] 李三华[1] 宋长伟[1] 覃成[1] 朱欣婷[1,4] 

机构地区：[1]遵义医学院贵州省普通高等学校特色药物肿瘤防治重点实验室,贵州遵义563000 [2]贵州省麻醉与器官保护基础研究重点实验室,贵州遵义563000 [3]遵义医学院附属口腔医院,贵州遵义563000 [4]遵义医学院基础医学院,贵州遵义563000

出　　处：《分析科学学报》2017年第4期541-544,共4页Journal of Analytical Science

基　　金：贵州省科技厅社会发展攻关项目(黔科合SY字[2011]3031);遵义医学院招标项目(F-614);贵州省科技厅;遵义市科技局;遵义医学院联合基金(黔科合J字LKZ[2013]31号);贵州省教育厅特色重点实验室建设项目(黔教合KY字[2014]212)

摘　　要：双向电泳(Two-dimensional Electrophoresis,2D)是蛋白质组学研究过程中的常用技术,而蛋白质样品的准确定量是做好双向电泳的前提条件。由于双向电泳的蛋白质样品在定量时存在试剂兼容性的问题,故需使用GE或Bio-rad公司研发的双向电泳专用蛋白质定量试剂盒,但此类试剂盒价格高昂且操作繁琐,因此亟待开发一种快速、准确、成本低廉的蛋白质定量方法。通过研究发现,蛋白质溶液与考马斯亮蓝-G250混合后,在近红外荧光成像系统680nm波长下可以被激发出荧光,且当蛋白质浓度在0.01~0.2mg·mL^(-1)范围内时,其荧光强度与蛋白质浓度具有很好的线性关系。基于此特点,建立了一种以96孔板为载体,利用近红外成像技术对蛋白质样品进行定量的方法,该方法可用于双向电泳前蛋白质样品的快速定量。Two-dimensional electrophoresis（2D）is a commonly used technique in proteomics study,and accurate quantification of protein is a precondition for 2Dexperiment.However,one of reagent used in 2Dexperiment is possibly incompatible with another during protein quantitation,and the special 2D protein quantitation kits developed by GE or Bio-rad companies are not only expensive but also time-consuming in operation.Our aim in the study is to explore a fast,accurate,and economical method for protein quantitation.Our results showed that protein-Coomassie brilliant blue（CBB）-G250 mixed solution could generate fluorescence when it is excited at 680 nm and the fluorescence intensity was strictly correlated with reference protein concentration（0.01-0.2 mg·mL^（-1））.Hence a near-infrared imaging method for protein quantitation performed in 96-well plates was developed,and the method presented here could be used for rapid protein quantitation before two-dimensional electrophoresis.

关 键 词：蛋白质定量 近红外成像 考马斯亮蓝 双向电泳 

分 类 号：O657.33[理学—分析化学]