Phospho-EGFR (pan), Phospho-EGFR (Tyr1068) & Total EGFR Cellular Assay Kits

HTRF® cell-based phospho and total-EGFR assay for anti-cancer research

Mutations that lead to an overexpression or hyperactivity of EGFR also known as ErbB1 or HER1, are associated with a number of cancers, such as lung, colorectal, prostate, glioblastoma cancer etc. This makes EGFR a key target for anti-cancer therapies immunoassay.

Cisbio's cell-based homogeneous phospho-EGFR assay kits are designed for the cell-based quantitative detection of phosphorylation on EGFR. One phospho kit allows the measurement of the specific EGFR phosphorylation on Tyr1068, while the other allows all tyrosine phosphorylations to be generically measured. The HTRF® total EGFR assay kit is designed for the quantitative detection of total EGFR, phosphorylated and unphosphorylated, to normalize the phosphorylation status of EGFR with respect to its steady-state level in cells.

The buffers of both HTRF® phospho- and total EGFR assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample.

   Check our lysis buffer compatibility      Check our species compatibility

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Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total EGFR assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-EGFR antibodies, one labeled with Cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with EGFR, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.
The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-EGFR can be quantitatively detected using the HTRF phospho-EGFR (Tyr1068), phospho-EGFR (pan) and total EGFR cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol – adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-well small volume assay plate where the HTRF reagents are added. This also enables monitoring the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

The protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol means enhanced speed and simplicity, enabling all throughputs and fast results while maintaining high quality and sensitive output.

Simplified Pathway

EGFR Epidermal growth factor receptor signaling

EGFR, the epidermal growth factor receptor, also known as HER1 or ErbB1 is a receptor tyrosine kinase and belongs to the ErbB family. EGFR is present on the cell surface with an extracellular ligand-binding domain, a hydrophobic transmembrane domain & a cytoplasmic region that contains a tyrosine kinase domain and a C-terminal tail with tyrosine residues. Binding of EGFR ligands, like the EGF family of peptide growth factors, e.g. EGF and TGF-α drives receptor homodimerization EGFR-EGFR or hetero-dimerization EGFR with another ErbB family member, leading to the activation of the EGFR tyrosine kinase domain and subsequently to auto- or trans-phosphorylation of specific tyrosine residues. The phosphorylated tyrosine residues in consequence provide docking sites for a variety of adaptor proteins, kinases & phosphatases, such as Shc, Grb2, PLCɣ & PI3K that induce downstream activation of several signal transduction cascades, principally the MAPK/ERK, PI3K/AKT pathways. Further inductions that may result from receptor activation are STAT signaling, Calcium/PKC signaling and nuclear localization of the receptor.

The transmitted signals from the EGF receptor via different pathways like AKT, ERK & STAT3 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, like lung, colorectal, prostate, glioblastoma etc., which makes EGFR a key target for anti-cancer therapies.

Product Performances

1. HTRF phospho-EGFR (Tyr1068) assay compared to Western Blot

Human A431 cells were grown in a T175 flask at 37 °C, 5% CO2 for 48 h. Cells were then stimulated with 100 nM EGF for 10 min. After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western-blot and by HTRF. By using HTRF phospho-EGFR (Tyr1068) only 100 cells are sufficient for minimal signal detection while 3,120 cells are needed for a Western Blot signal. The HTRF phospho-EGFR assay is at least 30-fold more sensitive than the Western Blot and shows optimal correlation.

2. HTRF total EGFR assay compared to Western Blot

A431 cells were grown in a T175 flask at 37 °C, 5% CO2 for 48h. Cells were then stimulated with 100 nM EGF for 10 min. After medium removal, the cells were lysed with 3mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western-blot and by HTRF. By using HTRF total-EGFR only 50 cells are sufficient for minimal signal detection while 780 cells are needed for a Western Blot signal. The HTRF total-EGFR assay is at least 15-fold more sensitive than the Western Blot and shows optimal correlation.

3.EGF dose-response on A431 cells

Human A431 cells (5,000 cells/well) were stimulated for 30 min at 37°C with various concentrations of EGF.

After a 30 min lysis incubation time, phosphorylated EGFR was measured using the one-plate assay protocol of the pan-EGFR assay.

4. Phospho-EGFR modulation in several human tumor cell lines

Human BxPC3, SKOV3, CRC1 and A431 cells were plated in 96 well plate and incubated for 24 h, at 37 °C-5% Co2. After 10 min incubation with increasing concentrations of agonist, medium was removed and cells were lysed with 50 µL of Lysis buffer for 30min at RT under gentle shaking.16 µL of lysate was transfered into a 384 sv white microplate and 4 µL of the HTRF phospho-EGFR detection reagents were added. HTRF signal was recorded after an overnight incubation.

Increasing concentration of EGF were added for 10 min on human pancreatic carcinoma BxPC3 cells plated at 50,000 cells per well.

 

Increasing concentration of TGF-α were added for 10 min on human epidermoid carcinoma A431 cells plated at 50,000 cells per well.

 

Increasing concentration of β-cellulin were added for 10 min on human ovarian carcinoma SKOV3 cells plated at various cell densities (25,000 - 50,000 – 100,000 cells per well)

 

Increasing concentration of EGF were added for 5 min on CRC1, human colorectal primary tumour cells plated at 50,000 cells per well

 

5. Inhibition of EGFR phosphorylation by intra cellular Tyrosine kinase inhibitors and extra cellular monoclonal antibodies

Cells were plated in 96-well plate and incubated for 24h, at 37 °C - 5% Co2, then, after a pre-treatment with a dose-response of inhibitors, the cells were stimulated with EGF at 37°C, 5% CO2. After these steps, medium was removed and cells were lysed with 50 µL of Lysis buffer for 30min at RT under gentle shaking.16µl of lysate was transferred into a 384-sv white microplate for detection and 4 µL of the HTRF phospho-EGFR detection reagents were added. HTRF signal was recorded after an overnight incubation. Results show the applicability of the HTRF phospho-EGFR assay to decipher the mechanism of action of small molecules and biologics targeting EGFR.

Inhibition measured on A431 cells with the Pan phospho-EGFR cellular kit

Cells were pre-treated with a dose-response of Erlotinib and Lapatinib, 2 small Tyrosine Kinase inhibitors. compound. A control was done using PQ401, a non-inhibitor compound. 

 

 

Cells were pre-treated with a dose-response of Cetuximab, a therapeutic monoclonal antibody. A control was done using a non-inhibitor MAb control (15441).

 

 

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.

 

 

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

 

 

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

assessment As expected, results obtained show a dose-response decrease of EGFR phosphorylation upon treatment with inhibitors while EGFR expression level remains constant

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
EGFR phospho-Y1068 kit - 500 tests64EG1PEG
EGFR phospho-Y1068 kit - 10,000 tests64EG1PEH
EGFR phospho-Y1068 kit - 50,000 tests64EG1PEY
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EGFR phospho-pan kit - 500 tests64HR1PEG
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EGFR phospho-pan kit - 10,000 tests64HR1PEH
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EGFR phospho-pan kit - 50,000 tests64HR1PEY
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EGFR total kit - 500 tests64NG1PEG
EGFR total kit - 10,000 tests64NG1PEH
EGFR total kit - 50,000 tests64NG1PEY
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Companion products

DescriptionCat. noProduct insertMSDS
EGFR phospho-Y1068 kit control lysate64EG1TDA
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EGFR phospho-pan kit control lysate64HR1TDA
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EGFR total kit control lysate64NG1TDA
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