Learn More
Thermo Scientific™ LanthaScreen™ ERK2 A375 Cell Line
Description
The Map Kinase (MapK) signal transduction cascade is activated by growth factors such as EGF, PDGF, and HGF. Binding of these factors to their respective cell surface receptors results in the initiation of receptor tyrosine kinase activity, which leads to the sequential phospho-activation of downstream kinases such as Ras, Raf, MEK, and Erk 1/2. Activated MEK phosphorylates Erk2 (Mapk1) proteins at a specific Thr-Tyr motif (Thr/Tyr 185/187). A number of constitutive active kinase mutants within the MAP kinase pathway have been implicated in oncogenesis. One of these mutants is BRAF(V600E), which is the most predominant oncogenic BRAF mutant. The human melanoma cell line A375 endogenously expresses BRAF(V600E), which leads to the constitutive activation of the MAP kinase pathway and phosphorylation of Erk2 in the absence of ligands.
LanthaScreenTM Erk2 A375 constitutively expresses GFP-Erk2 under control of a CMV promotor. Using this cell line, a homogenous immuno-assay has been developed in which the phosphorylation state of GFP-Erk2 is detected in cell lysates using a terbium-labeled anti-pTpY-185/187-Erk2 antibody, in a time-resolved FRET (TR-FRET) readout. This cell line can be used to evaluate compound activity against BRAF(V600E). GFP-Erk2 lentivirus was transduced into A375 cells, followed by selection with Blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as sorting marker. Using the lytic TR-FRET immuno-assay, this cell line is validated for EC50 and Z' under optimized conditions using EGF as a ligand for GFP-Erk2 phosphorylation. This assay has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance and cell lysis/equilibration time. Additional information using alternate stimuli and small molecule inhibitor is also provided.
Specifications
Specifications
| Antagonists | Raf Inhibitor I |
| Assay Entry | Cell-based terbium phospho-specific activity (stable) |
| Cell State | Dividing Cells |
| Content And Storage | LanthaScreen™ ERK2 A375 cells are shipped on dry ice. Store in liquid nitrogen immediately upon receipt, or thaw for immediate use. |
| Detection Method | Fluorescence |
| For Use With (Application) | TR-FRET |
| Gene Symbol | ERK2 |
| Primary Agonist | Constitutive |
| Product Line | LanthaScreen |
| Quantity | 1 Vial |
| Show More |
For Research Use Only. Not for use in diagnostic procedures.
By clicking Submit, you acknowledge that you may be contacted by Fisher Scientific in regards to the feedback you have provided in this form. We will not share your information for any other purposes. All contact information provided shall also be maintained in accordance with our Privacy Policy.