View the slide viewer

Have questions about this assay kit?

Human EGFR1 Reporter Assay Kit

SIZE SKU PRICE
1 x-96 well format assays
3 x-32 assays in-96 well format
1 x-384 well format assays
SIZE SKU
1 x-96 well format assays
3 x-32 assays in-96 well format
1 x-384 well format assays

Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Epidermal Growth Factor Receptor 1 (EGFR1). INDIGO’s EGFR1 Reporter Cells include the luciferase reporter gene functionally linked to a EGFR1 -responsive promoter. In addition to EGFR1 Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human EGFR1. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

  • Ready to Use Upon Receipt

  • Includes All Needed Components
  • Contains Transfected Reporter Cells
  • Eliminates Cell Licensing Fees
  • Clear, Reproducible Results
  • Consistent Results Lot to Lot

Product Specifications

Target TypeGrowth Factor Receptor
SpeciesHuman
Receptor FormNative
Assay ModeAntagonist
Kit Components
  • EGFR1 Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • EGF, (ref. activator; in PBS/0.1% BSA )
  • Detection Substrate
  • Detection Buffer
  • White, sterile, cell-culture ready assay plate
Shelf Life6 months
Orthologs AvailableNo
Shipping RequirementsDry Ice
Storage temperature-80C

Data

EGFR1 Agonist dose-response assays. EGFR1 activation assays were performed according to the protocol provided in this Technical Manual. 200 ul / well of EGFR1 Reporter Cell suspension was dispensed into the 96-well assay plate, which was then incubated for 4 hours. Concentrated stocks of the reference peptides EGF (provided), TGFb and Amphiregulin (Sigma) were prepared in PBS + 0.1% BSA, then further diluted using CSM to produce treatment media at the desired assay concentrations. The pre-culture media were discarded from the assay wells and 200 ul per well of respective treatment media were dispensed (n = 3/conc.), including ‘vehicle only’ control wells. ‘Mock’ reporter cells, which contain the STAT3-Luc reporter gene but lack expression of the EGFR1, were treated with EGF. Following a 22-hour incubation period treatment media were discarded, Luciferase Detection Reagent was added, and luminescence intensity per well was quantified. Values of average relative light units (RLU) and corresponding values of standard deviation (SD) and percent coefficient of variation derived from SD (%CV) were determined for each treatment condition. Non-linear regression analyses and EC50 calculations were performed using GraphPad Prism software. Plots show Fold Activation vs. Log10[ng/mL] for the various treatment materials; error bars depict +/- %CV.
200 µl / well of EGFR1 Reporter Cell suspension was dispensed into 96-well assay plates, which was then incubated for 4 hours. For inhibition-mode assays, prior to the end of the pre-culture period, CSM was supplemented with 1.4 ng EGF/ml (an approximate EC80 concentration). The EGF-supplemented CSM was then used to prepare the various treatment media. a.) The small molecule inhibitors Pelitinib and Gefitinib (Cayman Chem.), AG-1478, PD153035, and BIBU1361 (Tocris) were initially prepared in DMSO as 2 mM stock solutions that were further diluted using the EGF-supplemented CSM. Final assay concentrations began at 2 µM and proceeded with serial 5-fold decrements. Z’ values4 ranged between 0.77 and 0.84. b.) The antiEGFR antibody Cetuximab (MedChem Express; 5 mg/mL) was diluted using EGFsupplemented CSM to produce final assay concentrations beginning at 5 µg/mL and proceeding with serial 3-fold decrements. Pre-culture media were discarded and 200 µl/well of the prepared treatment media were dispensed (n = 3/conc.), including ‘vehicle only’ control wells. After 22 hr incubation treatment media were discarded, Luciferase Detection Reagent was added, and RLU/well were quantified. Plots are a.) RLU vs. Log10[nM], and b.) RLU vs. Log10[ng/mL] for the various test materials. Error bars depict +/- SD.

Target Background

EGFR1 is a single-pass transmembrane receptor, one of four members of the receptor tyrosine kinase (RTK) family. Binding interactions with extra-cellular signaling peptides such a epidermal growth factor (EGF), transforming growth factor alpha (TGFα), or amphiregulan lead to receptor dimerization and auto-phosphorylation and activation of associated intracellular signaling proteins. Interestingly, EGF Receptors demonstrate two alternative signal processing modes: one via the membrane-bound receptor and signaling at cell surface; and the other signaling through internalized receptors, nuclear translocation, and co-association with activated transcription factors to interact directly with target gene promoter sequences.

Activated EGF Receptors are known to signal through several different pathways, including those mediated by Ras, PI3K, PLC-γ, and JAK, culminating in the activation of specific transcription factors and the induction of respective target genes. Phosphorylation and activation of the transcription factor STAT3 is one prominent pathway utilized by EGFR1, and it is the signaling mechanism exploited by the reporter cells in the INDIGO kit.

INDIGO’s reporter cells contain the luciferase gene functionally linked to an upstream minimal promoter and tandem STAT3 genetic response element (GRE) sequences. Activated, dimeric STAT3P (or STAT3P associated with nuclear EGFR1P) bind the STAT3 GREs to initiate the formation of a complete transcription complex that drives Luc expression. Quantifying relative changes in luciferase activity in the treated reporter cells relative to the untreated cells provides a sensitive, dose-dependent surrogate measure of drug- or antibody-induced changes in EGFR1 activity.

Considering their significant role in rapid cell proliferation, and their involvement in the progression of many types of cancers, EGFR continues to command much interest as a target for the development of novel, specific, and predominantly inhibitory drugs and antibodies. Accordingly, the primary application of this EGFR1 Reporter Assay is to screen test materials for any functional activity, either agonist or inhibitory, that they may exert against the EGFR1.

Also available as a service

Epidermal Growth Factor Receptor 1 (EGFR1)

Have questions about this assay kit?