Analysis of Protein Kinase AKT with an AMMP™ Assay in Cell Culture and Tumour Lysates

In this application note by BioScale, inhibition of pSer473-Akt using both tissue culture and mouse xenografts is monitored. Activation of protein kinase AKT, through phosphorylation of both Thr308 and Ser473, promotes cell survival. A pair of commercially available monoclonal antibodies against both pSer473-Akt and pan-Akt was selected and each affinity tagged with either biotin or fluorescein. Using the ViBE™ Protein Analysis Platform with AMMP™ Assay technology, a standard curve was generated with in vitro phosphorylated (p473) recombinant Akt2. Using that derived standard curve a comparison of samples treated or untreated with PI3K inhibitors from either cell culture or xenograft tumor lysates was made using the ViBE™ BioAnalyzer with Li-Cor Western Blot analysis.