Sponsored by R&D Systems, Inc. Published in September 2010 2010 (p.3) DOI: 10.2144/000113522 Sponsored,vendor-submitted protocol
Cells are grown in 96-well plates and treated with the appropriate conditions, such as inhibitors or ligand stimulation. The cells are then fixed and permeabilized in the wells. This is followed by incubation with two primary antibodies derived from different species: a phospho-specific antibody and a normalization antibody that recognizes the total protein regardless of its phosphorylation status. Species-specific secondary antibodies labeled with horseradish peroxidase (HRP) and alkaline phosphatase (AP), and spectrally distinct fluorogenic substrates for each enzyme, are used for detection. The fluorescence of the phosphorylated protein is normalized to that of the total protein in each well to correct for well-to-well variations. Cell-Based ELISAs have been used to evaluate the effects of stimulators and inhibitors on cultured cells, and this has been accomplished with 10,000 cells, or less, per well. For example, phosphorylation of JNK (T183/Y185), Akt (S473), EGF R (Y1068), FRS2 (Y436), total protein levels of IkB-a induced by various stimuli, and the effects of kinase inhibitors, were assessed here using the Cell- Based ELISA method. The results were compared with Western blot and traditional sandwich ELISA. Once the cells are plated on 96-well microplates, the total hands-on time for the Cell-Based ELISA is approximately 3 hours, which is significantly less than other techniques. In addition, Cell-Based ELISAs are amenable to high-throughput applications and may prove a valuable addition to kinase inhibitor screening strategies.