R&D Systems Cell-Based ELISAs simultaneously measure the levels of two proteins in the same microplate well in whole, fixed cells, eliminating the need for lysate preparation. These simple, sensitive assays can be used with both adherent and non-adherent cells to analyze protein phosphorylation or assess total protein levels. Kits contain antibodies to the protein of interest and a second normalization protein. These two proteins are simultaneously detected in the same microplate well, thus, signals derived from the target protein can be normalized to that of the second protein, correcting for well-to-well variations and allowing target protein levels to be accurately assessed and compared across multiple samples. Cell-Based ELISAs are available in two different detection formats: fluorometric detection that is compatible with standard microplate fluorometers, and near-infrared detection that is compatible with the LI-COR® Odyssey® Infrared Imaging System.
Fluorometric Detection – Cell-Based ELISAs contain the components required to run an immunoassay using fluorogenic substrates, including two primary antibodies, species-specific secondary antibodies conjugated to either horseradish-peroxidase (HRP) or alkaline phosphatase (AP), and spectrally distinct fluorogenic substrates for each. Fluorescence is measured using a standard microplate fluorometer.
Near-infrared Detection - Cell-Based Infrared ELISAs contain the components required to run a near-infrared immunoassay, including two primary antibodies and species-specific secondary antibodies conjugated to spectrally distinct near-infrared fluorophores. Fluorescent signals are measured using an infrared imaging system, such as the LI-COR Odyssey Infrared Imaging System. Near-infrared fluorescence provides enhanced fluorescence stability and higher signal-to-noise ratios due to low background autofluorescence.
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|Figure 1. The MCF-7 human breast cancer cell line was untreated or treated with increasing concentrations of Recombinant Human IGF-I (Catalog # 291-G1) for 20 minutes. After fixation and permeabilization, levels of phosphorylated (S473) Akt were determined using either the Human/Mouse/Rat Phospho-Akt (S473) Pan Specific Cell-Based ELISA (Catalog # KCB887; purple bars) or the Human/Mouse/Rat Phospho-Akt (S473) Pan Specific Cell-Based Infrared ELISA (Catalog # KCBIR887; red bars) and normalized to total Akt protein levels in the same well to compensate for well-to-well differences. Values represent mean ± range of duplicate determinations.|
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