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For studying intracellular proteins involved in signaling, we offer several phosphoELISA™ kits for the measurement of total and phosphorylated, modified, or cleavage site-specific proteins. Our phosphoELISA™ kits have been designed to enable the specific and sensitive detection of phosphorylation of key signaling molecules. Advantages of our phosphoELISA™ kits include:
  • Specificity: two antibodies directed against an analyte provides better specificity than western blotting
  • Sensitivity: more sensitive than western blotting
  • Quantitation: get quantitative data in contrast to western blotting (Figure 1)
  • Fast results: 96-well format, results in about 4 hours, no densitometry analysis needed

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Featured PhosphoELISA™ Kit categories

Antibody Pair Kits

Matched, pre-titered, and fully optimized capture (coating) and detection antibodies allow you to build your own immunoassay.

Primary Antibodies

Search for primary antibodies against a wide range of markers, including signaling proteins, cell and organelle markers, cell junctions CD markers, and more.

Luminex® Assays

Bead-based technology enables the analysis of multiple targets in a single sample from a wide range of biological sources. Check our custom Luminex® assay panel.

Secondary Antibodies

Browse our secondary antibodies, with conjugates that include Alexa Fluor® and classic fluorescent dyes, HRP and AP, and more from a wide range of hosts.

PhosphoELISA™ kits

PhosphoELISA™ kits begin with a monoclonal antibody specific for a protein of interest (regardless of phosphorylation state) coated onto the wells of microtiter strips in a 96-well plate format.

Samples, including a standard containing protein of interest, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the protein antigen binds to the immobilized (capture) antibody, much like an immunoprecipitation.

After washing, a rabbit antibody specific for total protein or protein phosphorylated at a specific residue (e.g., Akt [pS473]) is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized protein captured during the first incubation.

After removal of excess detection antibody, a horseradish peroxidase-labeled anti-rabbit IgG (anti-rabbit IgG-HRP) is added. This binds to the detection antibody to complete the four-member sandwich.

After a third incubation and washing to remove all the excess anti-rabbit IgG-HRP, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of total or phosphorylated protein present in the original specimen. 

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Figure 1. Confirmation of western blotting data with quantitative results by phosphoELISA™ assay. (A) Quantitative data obtained using the STAT5a [pY694] Human ELISA Kit (Cat. No. KHO0761). (B) Western blotting results using a NuPAGE® Novex® gel (Cat. No. NP0321). Assays were performed in parallel.

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