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Novel approach for automated sequential immunoassay for quantitation and characterization of PI3K/AKT pathway proteins - Dr. Jessica Dermody

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The P13K/Akt signaling pathway modulates growth, survival and apoptosis and this pathway is frequently modulated in human cancers contributing to resistance to radiation and chemotherapy treatments. Akt is a target for specific inhibition and recently, a number of small molecules have been developed the pharmacologic properties of known inhibitors like wortmannin and LY294002. However, Pan-Akt inhibitors can result in unanticipated side effects due to the lack of specificity for Akt isoforms 1, 2 and 3. Therefore, detection and quantitation of Akt isoforms and their downstream targets for both expression levels and phosphorylation states is crucial for therapeutic drug development. Here we demonstrate application of ReplexTM to characterize the P13K/Akt signaling pathway. This approach uses sequential analysis of proteins separated and immobilized in a capillary, by performing either dual immune assays or immunoassay with total protein on the Simple Western platform chemiluminescense detection. Assays with control and LY294002 inhibitor- treated samples were developed. Proteins were first separated based on molecular weight based on capillary electrophoresis, followed by immobilization via UV-crosslinking. Next P13K/Akt pathway targets were sequentially probed in the same capillary with total and phospho-specific antibodies to determine the phosphorylated fraction relative to the total fraction. Primary antibodies from the first immunoprobe was removed with the detection probe with >95% efficiency, as confirmed by re-probing with the same secondary antibody. Target protein loss was negligible due to covalent immobilization to the capillary wall, which was confirmed with re-probing, thus validating the quantitative data generated using this sequential approach. In addition, total protein normalization was performed in tandem with the immunoassay in the same capillary. This approach enables normalization of phosphorylation levels and/or target abundance in cell line or tissue samples, correcting for change in protein content due to treatment, loading and/or other systemic errors. These results present the utility of the RePlex™ to quickly characterize and quantify proteins involved in signaling pathways targeted during development of cancer therapies.

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