Qi Xiaoying, Zhang Yunyun, Zhang Yuan, Ni Tianhui, Zhang Wenfeng, Yang Chunhua, Mi Jia, Zhang Jiandi, Tian Geng
Medicine and Pharmacy Research Center, Binzhou Medical University.
Yantai Zestern Biotechnique Co. LTD.
J Vis Exp. 2018 Aug 21(138):56885. doi: 10.3791/56885.
Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.
在细胞和组织水平上缺乏一种方便、定量、高通量的免疫印迹方法来绝对测定特定蛋白质的含量，这严重阻碍了蛋白质组学研究的进展。目前可用的免疫印迹技术所得结果也是相对的，这使得将独立研究与蛋白质样品的大规模分析相结合的任何努力都受到阻碍。在本研究中，我们展示了定量斑点印迹分析（QDB）的过程，以实现高通量形式的绝对定量。使用市售蛋白质标准品，我们能够确定从三种不同小鼠组织（肾脏、脾脏和前列腺）制备的蛋白质样品中帽状肌动蛋白样凝溶胶蛋白（CAPG）的绝对含量，并对实验细节进行详细解释。我们提出QDB分析作为一种方便、定量、高通量的免疫印迹方法，用于在细胞和组织水平上对单个蛋白质进行绝对定量。该方法将极大地有助于生物和生物医学研究各个领域中的生物标志物验证和途径验证。
