quantitative analysis of protein phosphorylations and interactions by multi-colour ip-fcm as an input for kinetic modelling of signalling networks定量分析蛋白质的磷酸化和交互的彩色ip-fcm作为输入信号网络的动力学模型.pdfVIP

Quantitative Analysis of Protein Phosphorylations and Interactions by Multi-Colour IP-FCM as an Input for Kinetic Modelling of Signalling Networks 1,2 3 ¨ 3 1,4,5 Sumit Deswal , Anna K. Schulze , Thomas Hofer , Wolfgang W. A. Schamel * 1 Max Planck Institute of Immunobiology and Epigenetics, and Faculty of Biology, Biology III, University of Freiburg, Freiburg, Germany, 2 Spemann Graduate School of Biology and Medicine, Freiburg, Germany, 3 Research Group Modeling of Biological Systems, German Cancer Research Center and BioQuant Center, Heidelberg, Germany, 4 BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany, 5 Centre of Chronic Immunodeficiency (CCI), University Medical Center Freiburg, and University of Freiburg, Freiburg, Germany Abstract Background: To understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success. Methodology/Principal Findings: We use multi-colour immunoprecipitation measured by flow cytometry (IP-FCM) for studying signal transduction events to unrivalled precision. In this method, antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence signals are measured by FCM. Combining th