近红外成像用于双向电泳前的蛋白质快速定量

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。

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

Two-dimensional electrophoresis(2D) is a commonly used technique in proteomics study,and accurate quantification of protein is a precondition for 2D experiment.However,one of reagent used in 2D experiment 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.