Phospho-STAT3 (Tyr705) cellular kit
All-in-one kit for robust detection of Phospho-STAT3 (Ser423/425)
Total EGFR cellular kit HTRF®
The total EGFR kit monitors cellular EGFR expression level and can be used as a normalization assay for the phospho-EGFR kits.
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Overview
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.
Benefits
- SPECIFICITY
- COMPATIBLE WITH MANY CELL TYPES
- DATA NORMALIZATION
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.
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.
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.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
Reagent solutions for PROTAC research
Our offer for PROTAC research summarized in this flyer - Flyers
PI3K/AKT/mTor translational control pathway - Posters
mechanism of action of EGFR inhibitors assessed in the pancreatic BXPC3 cell line - Posters
Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells
Analysis of a large panel of diverse biological samples and cellular models - Posters
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
HTRF phospho-assays reveal subtle drug-induced effects
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Unleash the potential of your phosphorylation research with HTRF
Unmatched ease of use, sensitivity and specificity assays - Videos
Product Insert EGFR total Kit / 64NG1PEG-64NG1PEH
64NG1PEG-64NG1PEH - Product Insert
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Safety Data Sheet EGFR total Kit / 64NG1PEG
64NG1PEG - Safety Data Sheet
Safety Data Sheet (DEU) EGFR total Kit / 64NG1PEG
64NG1PEG - Safety Data Sheet
Safety Data Sheet (ELL) EGFR total Kit / 64NG1PEG
64NG1PEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) EGFR total Kit / 64NG1PEG
64NG1PEG - Safety Data Sheet
Safety Data Sheet (ITA) EGFR total Kit / 64NG1PEG
64NG1PEG - Safety Data Sheet
Safety Data Sheet (SPA) EGFR total Kit / 64NG1PEG
64NG1PEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) EGFR total Kit / 64NG1PEG
64NG1PEG - Safety Data Sheet
Safety Data Sheet (ENG-US) EGFR total Kit / 64NG1PEG
64NG1PEG - Safety Data Sheet
Safety Data Sheet EGFR total Kit / 64NG1PEH
64NG1PEH - Safety Data Sheet
Safety Data Sheet (DEU) EGFR total Kit / 64NG1PEH
64NG1PEH - Safety Data Sheet
Safety Data Sheet (ELL) EGFR total Kit / 64NG1PEH
64NG1PEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) EGFR total Kit / 64NG1PEH
64NG1PEH - Safety Data Sheet
Safety Data Sheet (ITA) EGFR total Kit / 64NG1PEH
64NG1PEH - Safety Data Sheet
Safety Data Sheet (SPA) EGFR total Kit / 64NG1PEH
64NG1PEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) EGFR total Kit / 64NG1PEH
64NG1PEH - Safety Data Sheet
Safety Data Sheet (ENG-US) EGFR total Kit / 64NG1PEH
64NG1PEH - Safety Data Sheet
Safety Data Sheet (DEU) EGFR total Kit / 64NG1PEY
64NG1PEY - Safety Data Sheet
Safety Data Sheet (ELL) EGFR total Kit / 64NG1PEY
64NG1PEY - Safety Data Sheet
Safety Data Sheet (FRA-FR) EGFR total Kit / 64NG1PEY
64NG1PEY - Safety Data Sheet
Safety Data Sheet (ITA) EGFR total Kit / 64NG1PEY
64NG1PEY - Safety Data Sheet
Safety Data Sheet (SPA) EGFR total Kit / 64NG1PEY
64NG1PEY - Safety Data Sheet
Safety Data Sheet (ENG-GB) EGFR total Kit / 64NG1PEY
64NG1PEY - Safety Data Sheet
Safety Data Sheet (ENG-US) EGFR total Kit / 64NG1PEY
64NG1PEY - Safety Data Sheet
Plate Reader Requirement
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.
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