Application Of Phospho-Specific Antibodies In Reverse-Phase Protein Array

Reversed-phase protein array(RPPA)technology has become a powerful,sensitive,cost-effective,high-throughput technique platform for protein analysis in large number samples of tissues or cells.Through the combination of phospho-specific antibodies,RPPA has the ability to measure both total and phosphorylated proteins,and therefore represents a great powerful method to capture signal pathways and key signals in normal or diseased cells.RPPA is very suitable for the discovery of biomarkers,analysis of biopsy samples in clinical trials,pharmacodynamic assessment,key protein analysis affected by tumor targeted therapies.It is progressing to be a powerful tool in tumor sub-classification,patient prognosis,rational drug guidance,personalized treatment.Objective:Through preparing,characterizing and screening applicable and reliable phospho-specific antibody probesfor RPPA,and establishing cell signaling pathway patterns,these probes were used to explore signals of cell signaling pathway patternscombined with RPPA.This technology is expected to further apply in new drug development,clinical sample analysis and other fields.Methods: Phospho-specific antibodies against key kinases in Akt,MAPK and tyrosine kinase(RTK)pathways were prepared,and applications of these antibodies were verified by routine immunological methods.Then phospho-specific antibodies were further screened by RPPA to determine their applicability and reliability as probes.By using Akt,Erk/MAPK pathways in LNCa P cell which generate signal changes under 4 treatments(EGF,LY294,PMA,Na Cl),the cell signaling pathway patterns were established.Finally,screened phospho-specific antiobody probes were used in analysis of cell signaling pathway patterns combined with RPPA.Results: The project obtained 53 phospho-specific antibodies verified by western blotting,immunohistochemistry,immunofluorescence.The 25 antibody probes for RPPA with high applicability and reliability were obtained from screening.The 10 high quality antibody probes were applied to Akt,MAPK cell signaling pathway patterns analysis,and four groups data by RPPA assay were ultimately obtained.All 4 groups data 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 blotting which approved their validity and reliability.Conclusions:Phospho-specific antibody probes for RPPA with high applicability and reliability were successfully screened.The probes combined with RPPA were successfully used in the analysis of cell signaling pathway patterns.The results show that these probes are very suitable for the analysis of cell signaling pathway patterns in RPPA platform.The project research can effectively expand the RPPA antibody probes,and make it an advanced tool in the fields of new drug discovery,clinical samples analysis.