Zhu Zhongxin, Zhu Xinliang, Shen Jiayi, Zhou Ayi, Ni Maowei, Jin Litai, Cong Weitao
Zhejiang Provincial Key Laboratory of Biopharmaceuticals, Wenzhou Medical University, Wenzhou, Zhejiang, P. R. China; Wenzhou Undersun Biotechnology Co., Ltd, Wenzhou, Zhejiang, P. R. China.
Electrophoresis. 2015 Mar;36(6):924-9. doi: 10.1002/elps.201400404. Epub 2015 Feb 20.
A fluorescent quenching detection method for phosphoproteins in SDS-PAGE by using calconcarboxylic acid (CCA) was described. In this method, the fluorescence intensity of CCA was greatly increased with the presence of Al(3+) in the gel background, while in zones where phosphoproteins are located this intensity was absent because of fluorescence quenching phenomenon through the formation of CCA-Al(3+) -phosphoprotein appended complex. Approximately 4-8 ng of phosphoproteins can be selectively detected within 1 h (1D SDS-PAGE), which is similar to that of the most commonly used Pro-Q Diamond stain. The specificity of this novel technique for phosphoproteins was confirmed by dephosphorylation, Western blot, and LC-MS/MS analysis, respectively. Furthermore, to better understand the newly developed method, the detection mechanism of CCA stain was explored by fluorescent spectrometry. According to the results, it is believed that CCA stain may provide a new choice for selective, economical, MS compatible, and convenient visualization of gel-separated phosphoproteins.
描述了一种使用钙黄绿素（CCA）在SDS-PAGE中对磷蛋白进行荧光猝灭检测的方法。在该方法中，由于凝胶背景中Al(3+)的存在，CCA的荧光强度大大增加，而在磷蛋白所在区域，由于通过形成CCA-Al(3+)-磷蛋白附加复合物导致荧光猝灭现象，该区域不存在这种强度。在1小时内（一维SDS-PAGE）可选择性检测到约4-8 ng的磷蛋白，这与最常用的Pro-Q Diamond染色法相似。分别通过去磷酸化、蛋白质免疫印迹和液相色谱-串联质谱分析证实了这种新技术对磷蛋白的特异性。此外，为了更好地理解新开发的方法，通过荧光光谱法探索了CCA染色的检测机制。根据结果，认为CCA染色可能为凝胶分离的磷蛋白提供一种选择性、经济、与质谱兼容且方便的可视化新选择。
