A new fluorescent quenching method for the determination of phosphoproteins by using calconcarboxylic acid |
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| Authors: | Zhongxin Zhu Xinliang Zhu Jiayi Shen Ayi Zhou Maowei Ni Litai Jin Weitao Cong |
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| Affiliation: | 1. Zhejiang Provincial Key Laboratory of Biopharmaceuticals, Wenzhou Medical University, Wenzhou, Zhejiang, P. R. China;2. Wenzhou Undersun Biotechnology Co., Ltd, Wenzhou, Zhejiang, P. R. China;3. Zhejiang Cancer Hospital, Hangzhou, Zhejiang, P. R. China |
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| Abstract: | 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 Al3+ 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‐Al3+‐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. |
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| Keywords: | Calconcarboxylic acid Fluorescent quenching Phosphoprotein detection Pro‐Q Diamond |
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