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OR

Cell-Based ELISAs - Fluorometric Detection

R&D Systems Cell-Based ELISAs provide an alternative to traditional Western blot for the analysis of the relative levels of a specific protein in whole cells. Cell-Based ELISAs contain all the components required to run an immunoassay using fluorogenic substrates for the simultaneous detection of two proteins in whole, fixed cells without the need for lysate preparation. Kits are designed to use with either adherent or non-adherent cells for assessment of the relative levels of protein phosphorylation or total protein. Kits contain antibodies to the protein of interest and a second normalization protein. The two proteins are simultaneously detected in the same microplate well, permitting normalization of the signals derived from the target protein to that of the second protein to correct for well-to-well variations. These simple, sensitive assays are compatible with standard microplate fluorometers.

Features

  • No lysate preparation required
  • Results with as few as 10,000 cells per well
  • Measure two proteins simultaneously in the same well
  • Design allows for normalization of well-to-well variations
  • Complete kits require minimal optimization
  • Two detection formats available
  • Suitable for high-throughput analysis of non-adherent cells
  • Excellent alternative to Western blot and/or sandwich ELISA

Kit Contents

Assay Principle


View Full Assay Principle

Kit contents may vary. Please refer to product-specific inserts for a complete list of contents for individual assays.

  • Primary Antibody to Target Protein
  • Primary Antibody to Normalization Protein
  • HRP-conjugated Secondary Antibody
  • AP-conjugated Secondary Antibody
  • Substrate F1 specific for HRP
  • Substrate F2 specific for AP
  • Wash Buffer
  • Blocking Buffer
  • Plate Sealers
  • 96-well Clear-bottom Black Cell Culture Microplate
 

Detection of EGF R Phosphorylation (Y1068) in EGF-treated A431 Cells using the
Cell-Based ELISA.

Figure 1. The A431 human epithelial carcinoma cell line was untreated or treated with increasing concentrations of Recombinant Human EGF (Catalog # 236-EG) for 5 minutes. After fixation and permeabilization, EGF R phosphorylation (Y1068) levels were determined using the Human Phospho-EGF R (Y1068) Cell-Based ELISA (Catalog # KCB1095) and normalized to total EGF R in the same well to compensate for well-to-well differences (bar graph). Values represent mean ± range of duplicate determinations. Phosphorylated (Y1068) EGF R and total EGF R were also detected by Western blot (inset) using the antibodies supplied in the kit.