HTRF Human Total EGFR Detection Kit, 10,000 Assay Points

Total-EGFR assay principle

The Total-EGFR assay quantifies the expression level of EGFR in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-EGFR 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 EGFR 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 proteins expression under a no-wash assay format.

Total-EGFR 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 EGFR HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-EGFR 1-plate assay protocol

Detection of total EGFR 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.

Inhibition measured with Phospho-EGFR (tyr1068) and total EGFR cellular kits

Human epidermoid carcinoma A431 cells were pre-treated for 3 h with a dose-response of Cetuximab, an extracellular therapeutic antibody, then stimulated for 10 min with 200 nM EGF.

Inhibition measured with Phospho-EGFR (Tyr1068) and total EGFR cellular kits

Human pancreatic carcinoma BxPC3 cells were pre-treated for 30 min with a dose-response of ATCC528, an extracellular monoclonal antibody, then stimulated for 10 min with 200 nM EGF

Inhibition measured with Phospho-EGFR (tyr1068) and total EGFR cellular kits

Human epidermoid carcinoma A431 cells were pre-treated for 3h with a dose-response of Gefitinib, a small Tyrosine Kinase Inhibitor, then stimulated for 10 min with 200 nM EGF

EGFR epidermal growth factor receptor signaling simplified pathway

EGFR is a receptor tyrosine kinase that belongs to the ErbB family. Binding of EGFR ligands drive receptor homodimerization or hetero-dimerization leading to the activation of the EGFR tyrosine kinase domain and specific tyrosine residues.

The phosphorylated tyrosine residues then provide docking sites for a variety of factors that induce downstream activation of several signal transduction cascades.

The transmitted signals from the EGF receptor to the nucleus lead to the regulation of various biological functions such as cell proliferation, differentiation, survival, adhesion, migration & angiogenesis.

Mutations that lead to overexpression or hyperactivity of EGFR are associated with a number of cancers making EGFR a key target for anti-cancer therapies.