Hwang Sun-Young, Choi Jung-Kap
College of Pharmacy & Research Institute of Drug Development, Chonnam National University, Gwangju, South Korea.
Electrophoresis. 2017 Dec;38(24):3079-3085. doi: 10.1002/elps.201700243. Epub 2017 Sep 26.
Protein phosphorylation, one of the most important post-translational modifications, plays critical roles in many biological processes. Thus, it is necessary to precisely detect, identify and understand the phosphoproteins from protein mixture for the study of cell biology. We introduce a sensitive and specific detection method for phosphoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Anthracene Chrome Red A (ACRA) combined with the trivalent metal ion (Al ) is converted to fluorescent complex and the fluorescence is sharply increased by a change of pH environment. Phosphoproteins and non-phosphoproteins can be easily distinguished by the fluorescence quenching due to the structural change of ACRA-Al -phosphoprotein complex, unlike non-phosphoprotein complex. The method using ACRA is a negative staining based on the fluorescence quenching and has a high sensitivity comparable to Pro-Q Diamond stain. ACRA stain can detect 1-2 ng of α-casein and β-casein, 8-16 ng of ovalbumin (OVA) and κ-casein within 130 min. Moreover, the ACRA stain showed similar linear dynamic ranges and RSD to Pro-Q stain. The linear dynamic ranges of ACRA and the values of correlation coefficient were for OVA (8-500 ng, correlation coefficient r = 0.999), α-casein (4-500 ng, r = 0.992), β-casein (4-500 ng, r = 0.996), and κ-casein (8-500 ng, 0.998), respectively. On the other hand, the values of the relative standard deviations (RSD) ranged from 2.33 to 3.56% for ACRA. The method is sensitive, specific, simple, rapid and compatible with total protein stain such as SYPRO Ruby stain. Therefore, ACRA stain can be an advanced method for phosphoprotein detection in gels.
蛋白质磷酸化是最重要的翻译后修饰之一，在许多生物学过程中发挥着关键作用。因此，为了细胞生物学研究的需要，必须从蛋白质混合物中精确检测、鉴定和理解磷酸化蛋白。我们介绍了一种在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中灵敏且特异的磷酸化蛋白检测方法。蒽铬红A（ACRA）与三价金属离子（Al）结合形成荧光复合物，并且pH环境的变化会使其荧光急剧增强。与非磷酸化蛋白复合物不同，由于ACRA-Al-磷酸化蛋白复合物的结构变化导致荧光猝灭，从而可以轻松区分磷酸化蛋白和非磷酸化蛋白。使用ACRA的方法是基于荧光猝灭的负染色法，具有与Pro-Q Diamond染色相当的高灵敏度。ACRA染色可在130分钟内检测到1-2 ng的α-酪蛋白和β-酪蛋白、8-16 ng的卵清蛋白（OVA）和κ-酪蛋白。此外，ACRA染色与Pro-Q染色显示出相似的线性动态范围和相对标准偏差（RSD）。ACRA的线性动态范围和相关系数值分别为：OVA（8-500 ng，相关系数r = 0.999）、α-酪蛋白（4-500 ng，r = 0.992）、β-酪蛋白（4-500 ng，r = 0.996）和κ-酪蛋白（8-500 ng，0.998）。另一方面，ACRA的相对标准偏差（RSD）值在2.33%至3.56%之间。该方法灵敏、特异、简单、快速，并且与总蛋白染色如SYPRO Ruby染色兼容。因此，ACRA染色可以成为凝胶中磷酸化蛋白检测的一种先进方法。
