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Presented at 2015_EU

Determination of EGFR and VEGF Signaling Pathway Activity by Immuno-MALDI Targeting Akt1 and Akt2 in Colorectal Cancer Tumours
Robert Popp
UVic - Genome BC Proteomics Centre

Targeted treatment of colorectal cancer (CRC) only works in a minority of patients, and reliable methods to quantify signaling pathway activity are lacking. We therefore set out to develop immuno-MALDI (iMALDI) assays to determine expression levels and stoichiometry of critical phosphorylation sites in Akt1 (P31749) and Akt2 (P31751) in cancer cells and tumours. We quantified non-phosphorylated Akt1 from 100 µg protein of breast and CRC cells (MDA-231, SW480 and HCT116), and breast cancer tumours. Non-phosphorylated Akt2 has been quantified in 100 µg MDA-231 breast cancer cells. Recently, we improved sensitivity by 10-fold, which allowed decreasing the sample amount from 100 µg to 10 µg. The current lower limit of detection is 0.35 pg Akt1/µg lysate protein. We have developed iMALDI Akt1 and Akt2 assays for quantitation of non-phosphorylated Akt1 and Akt2.

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