考马斯亮蓝凝胶染色联合近红外成像用于双向电泳前的蛋白质定量  

Protein Quantitation in Pre Two-dimensional Electrophoresis by Near Infrared Imaging of Coomassie Brilliant Blue on Gels

作　　者：曹嵩[1,2] 邓文文[2] 朱欣婷[2] 胡姗姗[2] 喻田[1] 刘云[1,2] 

机构地区：[1]遵义医学院贵州省麻醉与器官保护基础研究重点实验室,遵义563099 [2]遵义医学院医学与生物学研究中心,遵义563099

出　　处：《中国细胞生物学学报》2013年第6期848-851,共4页Chinese Journal of Cell Biology

基　　金：卫生部卫生公益性行业科研专项(批准号:200802173);贵州省社会发展公关项目(批准号:黔科合SY字[2011]3031)资助的课题~~

摘　　要：双向电泳的蛋白质定量由于存在试剂兼容性的问题，常常需要使用GE或Bio-Rad公司专用的蛋白质定量试剂盒。这类试剂盒价格昂贵且实验操作繁琐，亟待建立一种准确、快速且廉价的双向电泳蛋白质样品的定量方法。不同浓度的蛋白质样品经十二烷基硫酸钠一聚丙烯酰胺凝胶电泳（SDS—PAGE）产生的蛋白质条带，经考马斯亮蓝（CBB）染色后能被近红外光激发出不同强度的荧光。利用这一性质，以牛血清白蛋白（BSA）作为蛋白质定量的标准品，使用0dyssey近红外荧光扫描成像系统绘制标准曲线用于双向电泳蛋白质样品的定量。实验结果表明，使用“700 channel”，在l～10μg的范围内，蛋白量与荧光强度表现出良好的线性和可重复性。因此，基于Odyssey近红外成像的考染凝胶定量的方法是一种精确快速可用于双向电泳蛋白质定量的方法。Because of the incompatibility of different protein quantitation reagents, special protein quantitation kits produced by GE or Bio-Rad with traditional methods are widely used in protein quantitation for twodimensional electrophoresis （2-D）. However, such kinds of kits are not only expensive but also time-consuming in operation. Hence an efficient but economical method for 2-D protein quantitation is badly needed. As a response to such need, the present research works on the theory that protein bands of diverse densities, first processed by SDSPAGE and then stained by Coomassie brilliant blue （CBB） G-250, can emit fluorescence with different intensities when scanned by near infrared. Specifically, Bovine serum albumin （BSA） was used as standard protein samples and Odyssey near infrared imaging system （Odyssey） was employed to draw curves to calculate the amount of protein for 2-D experiment. The results showed that within the range of 1-10 μg, the density of BSA protein was linear with the intensity of fluorescence when Odyssey was set at ＂700 channel＂. Moreover, the linear relationship is reproducible when retested. This suggests that our method, which is based on the near infrared imaging and CBB G-250 staining, is accurate, efficient and easily operating in protein quantitation.

关 键 词：蛋白定量 考马斯亮蓝染色 SDS-聚丙烯酰胺凝胶电泳 近红外成像 双向电泳 

分 类 号：Q51[生物学—生物化学]