Total-ERK assay principle

The Total-ERK assay quantifies the expression level of ERK in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-ERK assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of ERK in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total-ERK 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total ERK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-ERK 1-plate assay protocol

Detection of total ERK with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Validation of total-ERK1/2 assay on various cell lines

Human (Hela, A431, HEK293, Jurkat), murine (NIH 3T3) and Hamster (CHO-K1) cells were grown in a T175 flask at 37°C, 5% CO2 for 2 days. After removal of cell culture medium, 3 mL of supplemented lysis buffer 3 were added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging. ERK1/2 was detected using 16 µL of cell lysate. Cell lines from other species have not been tested, hence they must be evaluated case by case.

Total-ERK assay to control phosphorylation of ERK1/2

A431 cells (100,000 cells/well) were activated with EGF for 10 min, using the two-plate assay protocol of the Phospho-ERK1/2 and Total-ERK1/2 assays. As expected, results obtained show a dose-response increase of ERK1/2 phosphorylation upon EGF stimulation while ERK1/2 expression level remains constant.

MAPK/ERK cell signaling

The MAPK/ERK signaling cascade is activated by a wide variety of receptors involved in growth and differentiation. The core signal transduction cascade elicits regulation of numerous cellular processes including adhesion, migration, apoptosis, differentiation and metabolism. MEK1/2 catalyze the phosphorylation of ERK1/2 at Tyr204 and Thr202. In response, ERK phosphorylates hundreds of cytoplasmic and nuclear substrates. The wide complexity and diversity of MAPK signaling makes ERK a key regulator and major signaling node in biology.