Phospho-ERK1/2 (T202/Y204 + T185/Y187) + Total In-Cell ELISA Kit (Chemiluminescent) (ab207470)

Overview

  • Product name

    Phospho-ERK1/2 (T202/Y204 + T185/Y187) + Total In-Cell ELISA Kit (Chemiluminescent)
  • Detection method

    Luminescent
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

    Cell-based (quantitative)
  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Mouse, Rat, Chicken, Human
  • Product overview

    Phospho-ERK1/2 (T202/Y204 + T185/Y187) + Total In-Cell ELISA Kit (Chemiluminescent) (ab207470) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. The kit is designed specifically to quantify phosphorylated ERK1/2 and/or total ERK1/2. Cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are rapidly fixed to preserve activation-specific protein modifications. Each well is then incubated with a primary antibody that recognizes either phosphorylated ERK1/2 or total ERK1/2. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified chemiluminescent readout. The relative number of cells in each well is then determined using the provided Crystal Violet solution. The 96-well plate format is suitable for high-throughput screening applications.


    Phospho-ERK1/2 (T202/Y204 + T185/Y187) + Total In-Cell ELISA Kit (Chemiluminescent) (ab207470) contains 96-well plates and two primary antibodies. The phospho-ERK antibody was raised in rabbit against a synthetic phospho-peptide corresponding to residues Thr202 and Tyr204 of human ERK1 and Thr185 and Tyr187 of human ERK2. This antibody does not recognize un-phosphorylated ERK1 or ERK2, nor does it recognize other phosphorylated proteins. The total-ERK antibody recognizes both ERK1 and ERK2 regardless of their phosphorylation state. The kit can be used to study phosphorylated ERK1/2 relative to cell number or to determine ERK1/2 phosphorylation relative to the total ERK1/2 protein found in the cells. Once the phospho-ERK1/2 and total-ERK1/2 signals have been normalized for cell number, a comparison of the ratio of phosphorylated ERK1/2 to total ERK1/2 for each of the cell growth conditions can be made. The provided total-ERK1/2 antibody can be used as a positive control to demonstrate that the cells contain ERK1/2, the kit reagents are functional and that the protocol is performed correctly. Also, because fixed cells are stable for several weeks, many plates can be prepared simultaneously and then the assay performed when desired.

  • Notes

    Extracellular signal-regulated kinases 1 and 2 (ERK1/2), also called p44 and p42 MAP kinases (p44/p42), are members of the Mitogen Activated Protein Kinase (MAPK) family of serine/threonine protein kinases. Activated MAPKs phosphorylate specific serines and threonines on target protein substrates and function as late-stage messengers in signaling cascades, which convey external stimuli from the cell surface to cellular targets such as translation machinery, cytoskeletal proteins and transcription factors10.

    Signaling cascades that result in the activation of ERK1/2 can be initiated through a variety of receptors involved in growth and differentiation, including receptor tyrosine kinases (RTKs), integrins and ion channels12-14. Once activated, ERK1/2 can phosphorylate and regulate a variety of proteins. Cytosolic ERK1/2 targets include cytoskeletal proteins and kinases that regulate protein translation1, 15, 16. Activated ERK1/2 is also imported into the nucleus, where its phosphorylation targets include the transcription factors Elk-1, c-Jun, STAT1 and 3, Ets-1, c-Myc, ER, CREB and PPARg.

    The ERK1/2 cascade plays a crucial role in neuronal cells and has been studied extensively in recent years for its involvement in synaptic plasticity and memory function. For example, ERK has been shown to be potently activated by phosphorylation after a synaptically driven increase in intracellular calcium.  In addition, experiments performed on rats have shown that activation of ERK is required for memory consolidation of auditory fear conditioning. And, ERK has been shown to be essential for the maintenance of neuronal function and plasticity following traumatic brain injury.

    The Raf/MEK/ERK (MAPK) signal transduction cascade has been shown to regulate cell cycle progression and apoptosis in diverse cell types. Aberrant control of this cascade is often observed in transformed cell lines and is frequently linked with human cancers. ERK1/2 has also been shown to play an important role in oncogenicity and in the degree of progression within the mouse skin carcinogenesis system. Important apoptotic regulators modulated by ERK1/2 activity include Bad and Bcl-225.

  • Tested applications

    Suitable for: In-Cell ELISAmore details
  • Platform

    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab207470 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
In-Cell ELISA Use at an assay dependent concentration.

Protocols

References

ab207470 has not yet been referenced specifically in any publications.

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