BioTek HTRF AKT application note

AKT is a serine/threonine protein kinase that plays a key role in cellular processes. AKT has become a popular target for drug discovery campaigns, due to the fact that AKT inhibitors may help to treat a number of cancers. BioTek has demonstrated an automated homogeneous assay to probe AKT phosphorylation at its serine 473 residue using endogenous levels of kinase expression within human primary HUVEC cells.

The results showed that primary HUVEC provide a method to measure compound effects on important signalling pathways, providing increased data relevance compared to immortalised cell lines. The HTRF phospho-AKT (Ser473) assay effectively monitors PI-3 kinase and AKT cell signalling pathways in these cells. The assay may be automated using a 2-microplate protocol where pathway stimulation or inhibition is carried out in a 96-well microplate, then cell lysate aliquots are transferred to a 384-well microplate for assaying. The Synergy H4 reader detects signal changes caused by endogenous kinase phosphorylation events. Automation of the assay provides good robustness, and appropriate inhibition pharmacology is evident at the level of PI-3 kinase.

For the full application note, An automated Homogeneous Assay to Quantify Endogenous AKT

Phosphorylation in Human Umbilical Vein Endothelial Cells, click on link.