An AlphaScreen SureFire Phospho-ERK1/2 assay
Extracellular signal-regulated kinases (ERKs) are implicated in proliferation and cell growth pathways. They are part of the MAP Kinase pathway that is often deregulated in cancer cells. ERK 1/2 are activated by phosphorylation which either leads to phosphorylation of cytoplasmic substrates or to translocation into the nucleus where it activates transcription factors.
Here, phosphorylation of ERK1/2 in CHO cells was measured in 384 well format using the AlphaScreen SureFire kit. It employs two pERK specific antibodies that are coupled to an acceptor or donor bead. Excitation of the donor bead releases singulet oxygen. If the acceptor is in proximity of the donor, the singulet oxygen induces a cascade of reactions in the acceptor bead leading to emission of a light signal. Proximity of both beads only occurs if both bound to the analyte pERK. Therefore the emission light increases with increase ERK phosphorylation.