In-cell ELISAs for measuring relative total and phospho-EGFR levels.

The Thermo Scientific Pierce EGFR In-Cell ELISA Kits enable the relative quantitation of total and phosphorylated epidermal growth factor receptor (EGFR) in whole cells using a 96- or 384-well microplate format. The complete kits include a mouse monoclonal anti-EGFR antibody, rabbit monoclonal anti-phospho-(Y1173) EGFR antibody, detection reagents and all buffers required for either colorimetric or near infrared detection.

 

Highlights of the In-Cell ELISA Kits:

• Two reliable detection formats available: (Learn more)
  • colorimetric detection kit for use with standard microplate readers
  • near infrared fluorescent detection kit for use with IR imaging system
• Whole-cell detection requires no cell lysis, protein extraction or special sample preparation
• Provides better data and higher throughput than quantitative Western blotting
• Less costly (one well vs. one lane) than Western blotting

 

Overview of EGFR and phosphorylation of EGFR:

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein and receptor tyrosine kinase and plays a vital role in the regulating many of the main cellular signal transduction pathways. Binding of EGFR by ligands like epidermal growth factor (EGF) and transforming growth factor (TGF) alpha cause conformational changes and activation of EGFR tyrosine kinase activity through receptor dimerization and autophosphorylation. Tyr 1068, Tyr 1148, and Tyr 1173 are the major sites of EGF-dependent autophosphorylation. The phosphotyrosines on EGFR mediate signal transduction through interaction with signaling proteins like of Grb2 and SHC. Phosphorylation of Tyr 1173 on EGFR mainly regulates signaling through the MAP kinase pathway. The EGFR In-Cell ELISA Kit enables simultaneous detection of both total and phospho-(Y1173) EGFR.

 

Thermo Scientific Pierce EGFR In-Cell ELISA Kits give similar results with near IR and colorimetric detection methods. Both the near infrared and colorimetric detection formats for the EGFR In-Cell ELISA Kits show similar fold-induction in EGF stimulated A431. Z-factor (0.82 and 0.80, respectively) indicate comparable and robust performance between the near IR and colorimetric formats.

 

The In-Cell ELISA Kit provides a fast and reliable format for determination of EGFR protein levels, activation and inhibition. Evaluation of dose-dependent activation and inhibition of EGFR in A431 cells with EGF and PD169393. Panels A and B show an increase in phospho-EGFR with increasing doses of EGF. The EGF-induced phosphorylation of EGFR is inhibited with increasing concentrations of PD169393 (panel C, rows C and D), whereas total EGFR does not significantly change with either treatment. No significant staining is seen in the absence of primary antibodies (panel A, column 1 and panel C, column 12). Panels B and D show that the fluorescence (RIU, left Y-axis) values obtained with the near infrared detection method can be overlaid on top of the data obtained with the colorimetric detection method (right Y-axis).

 

Related Products and Links:
ELISA reagent reservoirs and sealing tape
Browse all In-Cell ELISA Kits for specific targets
Basic In-Cell ELISA Detection Kits – basic kit to be used with your primary antibodies

Product #	Description	Pkg. Size
62205	EGFR Colorimetric In-Cell ELISA Kit Sufficient materials for 1 x 96 wells Includes: Blocking Buffer, 50 ml 20X Tris Buffered Saline, 50 ml Surfact-Amps 20 (10% Tween-20 Detergent), 10 ml Surfact-Amps X-100 (10% Triton X-100 Detergent), 10 ml HRP Conjugate, 0.14 ml TMB Substrate, 58 ml TMB Stop Solution, 55 ml Janus Green Whole-Cell Stain, 50 ml Elution Buffer, 2 x 30 ml Thin Plate Seal Assembly, 8 each Anti-Phospho EGFR (Y1173) Antibody Anti-EGFR Antibody	kit
62210	EGFR Near Infrared In-Cell ELISA Kit Sufficient materials for 1 x 96 wells Includes: Blocking Buffer, 100 ml 20X Tris Buffered Saline, 50 ml Surfact-Amps 20 (10% Tween-20 Detergent), 10 ml Surfact-Amps X-100 (10% Triton X-100 Detergent), 10 ml DyLight 680 Goat Anti-Mouse IgG (H+L), 55 µl DyLight 800 Goat Anti-Rabbit IgG (H+L), 55 µl Thin Plate Seal Assembly, 8 each Anti-Phospho EGFR (Y1173) Antibody Anti-EGFR Antibody	kit