Sharma Renu, Sharma Amit, Kumar Atul, Jaganathan Bithiah Grace
Stem Cells and Cancer Biology Group, Department of Biosciences and Bioengineering, Guwahati, India.
Bio Protoc. 2019 Oct 20;9(20):e3395. doi: 10.21769/BioProtoc.3395.
Protein phosphorylation is one of the most important post-translational modifications, which acts as a reversible on or off switch for the activity of a large number of proteins. Analyzing the phosphorylation status of different proteins can reveal the alterations in the state of the cells in response to cellular damage, cancer and pharmaceutical drugs. Techniques such as mass spectrometry, radiolabeling, 2D-gel electrophoresis and western blotting are used to quantify protein phosphorylation. These assays can quantify phosphorylation in the bulk population of cells, however, flow cytometry can couple cell surface marker expression data with phosphorylation data to understand differential signaling in a sub-population within a heterogeneous population of cells. Our protocol describes the use of flow-cytometry for rapid and single cell-based quantification of intracellular phospho-protein with the help of anti-phospho protein specific antibody.
蛋白质磷酸化是最重要的翻译后修饰之一，它作为大量蛋白质活性的可逆开启或关闭开关。分析不同蛋白质的磷酸化状态可以揭示细胞状态在响应细胞损伤、癌症和药物时的变化。诸如质谱分析、放射性标记、二维凝胶电泳和蛋白质印迹法等技术被用于量化蛋白质磷酸化。这些测定法可以量化细胞总体中的磷酸化情况，然而，流式细胞术可以将细胞表面标志物表达数据与磷酸化数据相结合，以了解异质性细胞群体中一个亚群内的差异信号传导。我们的方案描述了借助抗磷酸化蛋白特异性抗体，使用流式细胞术对细胞内磷酸化蛋白进行快速且基于单细胞的量化。
