Thursday, March 21, 2013

Applications Thursday: ERK 1/2 phosphorylation using AlphaScreen SureFire

Activation of of ERK1/2 is central to a number of signaling pathways that convert extra-cellular signals that bind to receptors into cellular responses such as changes in transcription/translation. As a result, monitoring the phosphorylation status of ERK1/2 as an indication of activation can be used as a read out of receptor function and is therefore a useful tool in screening of compounds that alter receptor function.

AlphaScreen measurement of activated
Gi and Gq coupled GPGR's
Analysis of ERK phosphorylation can be done in a high-throughput format using the AlphaScreen  SureFire Phopho-ERK1/2 assay which is based, in principle, on an immuno-sandwich assay. The result is that when ERK1/2 is phosphorylated a donor bead and an acceptor bead are brought into close proximity. Laser excitation of the donor converts ambient oxygen to singlet oxygen initiating a cascade of chemical reactions that result in a luminescent signal in the acceptor bead.

In application note 210 from BMG LABTECH this approach was used to produce dose response curves for Gi and Gq G-protein coupled receptor agonists in a 384 well format using the POLARstar Omega. Although the POLARstar Omega does not have a laser for excitation, the use of AlphaScreen optimized filters allow this microplate reader to consistently attain sensitive results.

For more information on this and other applications for all of the microplate readers that BMG LABTECH has to offer please visit the applications center on our web site.