Phosphorylation ELISA Kits

RayBio® Phosphorylation ELISA kits are a rapid, sensitive, high-throughput, cost-effective and convenient methods for monitoring the activation of important biological pathways in cells or tissues. Unlike immunoblots, which require several days of processing to obtain parallel signals for phosphorylated and total target protein, the Raybiotech ELISA method achieves competing sensitivity within a 6 hour processing time.

The Phosphorylation ELISA is an in vitro enzyme-linked immunosorbent assay for measuring phosphorylated protein in cell lysate samples. A specific capture antibody (anti-pan-antibody or anti-phosphorylated antibody) has been coated onto a 96-well plate. Samples are pipetted into the wells and target proteins present in a sample are bound to the wells by the immobilized antibody. The wells are washed and a anti-phosphorylated antibody or anti-pan-antibody is used to detect the target protein. After washing away unbound antibodies, secondary antibodies are pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of target bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm

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How It Works

Phosphorylation ELISAs utilize the sandwich-based design principle for specific measurement of phosphorylated and total protein.

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Recombinant Human EGF Stimulation of A431 Cell Lines

A431 cells were treated or untreated with 100 ng/ml recombinant human EGF for 10 min. Cell lysates were analyzed using this phosphoELISA and Western Blot.

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Sensitivity

The A431 cells were treated with 100 ng/mL recombinant human EGF for 20 minutes to induce phosphorylation of EGF R. Serial dilutions of lysates were analyzed in this ELISA and by Western blot. Immunoblots were incubated with anti-phospho-EGFR (Tyr 1086).