Cell-Based Infrared ELISAs
R&D Systems Cell-Based Infrared ELISAs provide an alternative to traditional Western blot for the analysis of the relative levels of a specific protein in whole cells. Cell-Based Infrared ELISAs contain all the components required to run a near-infrared immunoassay to simultaneously measure the levels of two proteins in the same microplate well in whole, fixed, adherent or non-adherent cells without the need for lysate preparation. The kits utilize LI-COR® IRDye® 800CW-conjugated and IRDye® 680LT-conjugated secondary antibodies, which provide enhanced signal sensitivity due to low background autofluorescence in the near-infrared region, and are compatible with infrared imaging systems, such as the LI-COR Odyssey® Infrared Imaging System. Cell-Based Infrared ELISAs come in two formats: phospho-protein kits contain antibodies to measure both the phosphorylated and the total protein, while total protein kits contain antibodies to the protein of interest and a second housekeeping protein. Thus, both formats allow for the accurate comparison of normalized target protein levels across different wells.
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
- IRDye® 800CW-conjugated Secondary Antibody*
- IRDye® 680LT-conjugated Secondary Antibody*
- Wash Buffer
- Blocking Buffer
- Plate Sealers
- 96-well Clear-bottom Black Cell Culture Microplate
Detection of Akt Phosphorylation (S473) in
IGF-I-treated MCF-7 Cells using the Cell-Based Infrared ELISA.
Figure 2A. 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, phosphorylated (S473) Akt and total Akt protein levels were determined using the Human/Mouse/Rat Phospho-Akt (S473) Pan Specific Cell-Based Infrared ELISA (Catalog # KCBIR887).
Figure 2B. Histogram profiles of the levels of phosphorylated (S473) Akt, normalized to total Akt protein levels in the same well, were generated by analysis of fluorescence intensities. Values represent mean ± range of duplicate determinations. Phosphorylated (S473) Akt and total Akt were also detected by Western blot (inset) using the antibodies supplied in the kit.
LI-COR, Odyssey, and IRDye are registered trademarks of LI-COR, Inc.
*This product is covered by US 6,995,274; US 7,504,089; foreign equivalents and patents pending.