SureFire Cellular Assay
TGR BioSciences (www.tgr-biosciences.com.au) recently developed the SureFire cellular ERK 1/2 assay. This assay is capable of detecting phosphorylation of ERK 1/2 in a cell-based format and allows the detection of not only Gq/11-coupled receptor activation but also many of the more difficult to detect Gi/o-coupled receptors and even some Gs-coupled receptors.
The naturally high intracellular concentration of ERK 1/2 means that no transfections, dye loading, or pre-stimulations are required to perform the assay. This, in turn, permits the generation of cell-based assay data that has a high degree of physiological relevance and allows measurement of ERK 1/2 activation in cells by endogenous receptors, including primary cells.
Because the assay is homogeneous and amenable to automation, the SureFire cellular ERK 1/2 assay can be used not only in lab-scale experiments but also in high-throughput screens performed by the pharmaceutical industry.
The SureFire cellular ERK 1/2 assay can be performed on cells grown in either 96-, 384-, or 1536-well microtitre plates. Generally, cultured cells are stimulated with the appropriate agonist for an optimal time, usually 515 minutes, and lysed with SureFire Lysis buffer.
For inhibition experiments, a similar protocol is followed, but the cells are pre-incubated with the inhibitor prior to stimulation. To determine the extent of ERK 1/2 phosphorylation, the lysed samples are simply activated' with SureFire Activation buffer and incubated with SureFire Reaction buffer containing AlphaScreen beads (PerkinElmer) for two hours, at room temperature.
The signal in the wells can then be measured using a plate reader, capable of AlphaScreen measurements (Figure 2). There are no washing steps required and a minimum of liquid handling, affording easy automation and reduced handling errors.