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A new fluorescent quenching method for the determination of phosphoproteins by using calconcarboxylic acid

Author:
Zhu, Zhongxin, Zhu, Xinliang, Shen, Jiayi, Zhou, Ayi, Ni, Maowei, Jin, Litai, Cong, Weitao
Source:
Electrophoresis 2015 v.36 no.6 pp. 924-929
ISSN:
0173-0835
Subject:
Western blotting, dephosphorylation, fluorescence, gels, phosphoproteins, polyacrylamide gel electrophoresis, spectroscopy
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.
Agid:
1323254