“PhosphoELISA” kits are phospho-specific sandwich ELISA (enzyme-linked immunosorbent assay) kits that allow for quantitative measurement of protein inside the cell. Phospho-specific ELISA kits can be used during signaling studies to measure phosphorylation, modification, cleavage site-specific and total proteins. For more information visit our Phospho-Specific ELISA Kit page.
We offer a large menu of Thermo Scientific Pierce and Novex phospho-specific kits including STAT3, CREB, PRAS40, p38, AMPK alpha, PTK2 and Caspase 3 signaling. These kits are validated and manufactured to help ensure excellent quality and reproducibility. The kits meet quality-controlled specifications for criteria such as sensitivity, dynamic range, precision, specificity, recovery and lot-to-lot consistency. See our ELISA Selection Tool to find a kit to help with your research needs.
The ELISA method is a benchmark for quantitation of antigens. ELISAs are adaptable to high-throughput screening because results are rapid, consistent and relatively easy to analyze. The best results have been obtained with the sandwich format, utilizing highly purified, pre-matched capture and detection antibodies. The resulting signal provides data which is very sensitive and highly specific.
Phospho-specific protocols (Figure 1) begin with a capture antibody, specific for a protein of interest, coated onto the wells of microplates. Samples, including a standard containing protein of interest, control specimens, and unknowns, are pipetted into wells. During the first incubation, the protein antigen binds to the capture antibody. After washing, a detection antibody is added to the wells, this antibody binds to the immobilized protein captured during the first incubation. After removal of excess detection antibody, an enzyme-linked (i.e., HRP) secondary antibody is added and binds to the detection antibody. After a third incubation and washing to remove the excess secondary antibody, a substrate solution is added and is converted by the enzyme to a detectable form (color signal.) The intensity of this colored product is directly proportional to the concentration of antigen present in the original specimen. Thermo Scientific Pierce and Novex phospho-specific kits have been designed to enable specific detection of phosphorylation, offer great specificity and correlate well to western blot data.
Figure 1. Scheme of a typical phospho-specific protocol. Our ready-to-use Phospho-specific ELISA kits come with standard, detection antibody, HRP conjugate, chromogenic substrate and stop solution to complete all steps listed above.
Note: The protocol provided is representative of most phospho-specific ready-to-use ELISA kits. Protocols for individual kits may differ. Depending on the protein of interest, antibodies, buffers, or substrates being used, this general protocol may need to be optimized. For more information see Overview of ELISA and ELISA Development and Optimization.
Run time: 4 hours — 30 minutes hands-on time.
Note: A standard curve must be run with each assay for quantification.
Note: If using an Antibody Pair Kit, to build your own ELISA, you will need to coat the capture antibody on to a 96-well microplate yourself. For this process you will need: a 96-well microplate, capture antibody and blocking buffer (usually BSA or milk diluted in PBS). You will follow these steps before starting the above protocol:
Thermo Scientific Pierce and Novex phospho-specific ELISA kits are highly tested and validated. Below is data from our most popular kits. For more information about our range of phospho-specific ELISA kits please visit our Phospho-Specific ELISA Kit page.
|Figure 2. ERK 1/2 [pT185/pY187] Phospho-ELISA Kit, Human. Using the above protocol samples were serially diluted and run using the ERK 1/2 [pT185/pY187] Phospho-ELISA Kit, Human (Cat. No. KHO0091). Parallelism between ERK 1/2 standard and PMA-treated Jurkat (immortalized human T lymphocyte) cell lysate was demonstrated.
|Figure 3. PRAS40 [pT246] Phospho-ELISA Kit, Human. Using the above protocol samples were serially diluted and run using the PRAS40 [pT246] Phospho-ELISA Kit (Cat. No. KHO0421). Parallelism between PRAS40 standard and Jurkat (immortalized human T lymphocyte) cell lysate was demonstrated.
For Research Use Only. Not for use in diagnostic procedures.