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Preparation of Phospho-Specific Antibodies to Explore Signals of Cell Signaling Pathway Patterns through Reverse-Phase Protein Array
Lin Shikang1,2*, Sun Boqing2, Wang Qiwei1, Hu Yunyong2, Gu Ning1
1School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China; 2Nanjing Chuanbo Biotech. Co., Ltd., Nanjing 210031, China
Abstract: Phospho-specific antibodies as probes with reliability for application in reverse-phase protein array (RPPA) were prepared， characterized and screened. Combined with RPPA， they were used to explore signals of cell signaling pathway patterns. The cell signaling pathway patterns were established using Akt， Erk/MAPK pathways in LNCaP cells which generate signal changes under 4 treatment methods (EGF， LY294， PMA， NaCl). 4 groups of patterns data by RPPA assay were ultimately obtained， as well as 25 phospho-specific antibodies probes with high availability for RPPA. 4 groups data all showed dynamic changes of the phosphorylation status of key signaling moleculars in the pathways which responded for cell external treatments. The RPPA assay data were consistent with the results of Western blot which approved the validity and reliability of these data. The results showed that these phospho-specific antibody probes combined with the RPPA platform were very suitable for the analysis of cell signaling pathway patterns. This method can effectively expand the RPPA antibody probes， and make it an advanced tool in the fields of new drug discovery， clinical samples analysis etc.
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