关键词：蛋白质磷酸化；双色红外荧光；化学发光

    摘要：目的 通过与化学发光法比较，了解双色红外荧光技术在目标蛋白磷酸化检测中的优势。 方法 分别取内毒素休克小鼠不同时间肺组织，制备组织匀浆蛋白样品。用SDS-PAGE电泳分离后转膜，将膜分别与磷酸化的或/和总的p42/44 MAPK抗体孵育，二抗用耦联有AlexaFluor 680和IRdye 800的荧光抗体或辣根过氧化物酶抗体，通过双色红外荧光系统和化学发光法进行蛋白质磷酸化检测。结果 红外荧光检测技术和化学发光检测结果均显示内毒素处理后小鼠肺组织p42/44 MAPK磷酸化水平发生了改变，1 h最高，12 h以后逐渐恢复到接近正常水平。两种方法检测结果显示了较好的一致性，相比之下双色红外荧光技术具有更高的灵敏度，操作方便，节省时间以及可以同时检测两种蛋白等优点。 结论 双色红外荧光技术在蛋白磷酸化的检测方面具有较好的应用前景。

    Application of two-color infrared fluorescence for detection of phosphorylation of protein

    LIU Jing-hua¹, TANG Jing¹, LAN Xing-guo¹, LIU Ya-wei¹, LI Zhi-jie¹, CHENG Fang², DENG Peng¹, JIANG Yong<sup>1

    1</sup>Department of Pathophysiology and Key Laboratory of Functional Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, China; ²Gene Limited Company, Shanghai, 200233, China

    Abstract: Objective To assess the value of two-color infrared fluorescence imaging system in detecting protein phosphorylation in comparison with chemiluminescent detection. Method The lung tissue homogenate of mice treated with lipopolysaccharide (LPS) for different time were prepared to separate the proteins by SDS-polyacrylamide gel electrophoresis followed by transfer of the proteins onto PVDF membranes. The membranes were incubated with the antibodies against total p42/44 MAPK/phospho-p42/44 MAPK, and then with goat anti-mouse or anti-rabbit secondary antibodies conjugated to AlexaFluor 680, IRdye 800 or horseradish peroxidase. The blotted proteins were detected and quantified using Odyssey infrared imaging system or chemiluminescent detection. Results LPS treatment rapidly induced p42/44 MAPK phosphorylation, which reached the peak levels 1 h after the treatment and resumed the baseline level at 12 h. Consistent results were obtained by the two detection methods, but two-color infrared fluorescence imaging system showed better sensitivity in detecting the target protein, and was easy to manipulate with good efficiency and capable of analyzing two proteins simultaneously. Conclusion Two-color infrared fluorescence system is a powerful system for detecting phosphorylation of proteins. ......