Skip to main content

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

The total EGFR kit monitors cellular EGFR expression level and can be used as a normalization assay for the phospho-EGFR kits.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Part Number: 64NG1PEG
Unit Size:
Part Number: 64NG1PEH
Unit Size:
  • The Total EGFR cellular assay kit monitors total EGFR and is used as a normalization assay with our phospho-EGFR kits. 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 therapy-related immunoassay.

  • Assay Points
    10000
    Assay Target Type
    Kit
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Therapeutic Area
    Oncology & Inflammation
  • 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 protein’s expression under a no-wash assay format.

    phospho-how-it-works-total-egfr-assay-principle

     

    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.

    phospho-how-it-works-total-egfr-2-plate-assay-protocol

     

    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.

    phospho-how-it-works-total-egfr-1-plate-protocol

     

  • HTRF total EGFR assay compared to WB

    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.

    assay-validation-egfr-total-1

     

    assay-validation-egfr-total-2

     

    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.

    assay-validation-egfr-total-3

     

    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

    assay-validation-egfr-total-4

     

    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

    assay-validation-egfr-total-5

     

  • 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.

    phospho-pathway-egfr-64ng1peg

     

Resources

1-1 of 1 Resources
Guide
HTRF solutions, guide to major applications
htrf, guide, application, autoimmunity, cardiology, assays