Overview

  • Product name

    Phospho-MEK1/2 (S217/S221) + Total In-Cell ELISA Kit
    See all MEK1 + MEK2 kits
  • Detection method

    Colorimetric
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

    Cell-based (quantitative)
  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Product overview

    Phospho-MEK1/2 (S217/S221) + Total In-Cell ELISA Kit (ab207479) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. The kit is designed specifically to quantify activated (phosphorylated) MEK1/2 and/or total MEK1/2. 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 MEK1/2 or total MEK1/2. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified colorimetric 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.


    The Phospho-MEK1/2 (S217/S221) + Total In-Cell ELISA Kit (ab207479) contains 96-well plates and two primary antibodies. The phospho-MEK1/2 antibody was raised against a synthetic phospho-peptide corresponding to residues surrounding Ser217 and Ser221 of human MEK1/2. This antibody recognizes MEK1/2 only when phosphorylated at S217/221, and also reacts with MEK1/2 when singly phosphorylated at Ser217 but not at Ser221. It does not cross-react with related kinases. The total-MEK1/2 antibody recognizes MEK1/2 proteins regardless of the phosphorylation state.


    The kit can be used to study phosphorylated MEK1/2 relative to cell number or to determine MEK1/2 phosphorylation relative to the total MEK1/2 protein found in the cells. Once the phospho-MEK1/2 and total-MEK1/2 signals have been normalized for cell number, a comparison of the ratio of phosphorylated MEK1/2 to total MEK1/2 for each of the cell growth conditions can be made.

  • Notes

    MEK1 and MEK2 are located upstream of the MAP kinase ERK1/2, which can only be activated by MEK. Raf phosphorylates and thus activates MEK1/2 through phosphorylation of two serine residues at positions 217 and 221, which are located in the activation loop of subdomain VIII. Activated MEK then phosphorylates ERK1/2 on both a tyrosine and threonine residue. Activation of ERK leads to activation of other downstream kinases such as the p90rsk and MAPKAP, as well as several transcription factors including Elk-1, Jun and c-Myc. 

    MEK participates in a wide range of cellular processes including cell proliferation, differentiation and apoptosis. Constitutive activation of MEK1/2 results in cellular transformation. While MEK has not been identified as an oncogene product, it is the focal point of many signal transduction mitogenic pathways activated by proven oncogenes. This protein kinase has been reported to be a likely target for pharmacologic intervention in proliferative diseases.

  • Tested applications

    Suitable for: In-Cell ELISAmore details
  • Platform

    Microplate

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    1% SDS Solution 1 x 22ml 5 x 22ml
    10% Triton X-100 1 x 10ml 5 x 10ml
    10X PBS 1 x 120ml 5 x 120ml
    1X Antibody Blocking Buffer 1 x 22ml 5 x 22ml
    1X Antibody Dilution Buffer 1 x 30ml 5 x 30ml
    96-well tissue culture plate 2 units 10 units
    Anti-rabbit HRP-conjugated IgG 1 x 11µl 5 x 11µl
    Crystal Violet Solution 1 x 22ml 5 x 22ml
    Developing Solution 2 x 11ml 10 x 11ml
    Phospho-MEK1/2 antibody 1 x 9µl 5 x 9µl
    Plate sealer 2 units 10 units
    Stop Solution 2 x 11ml 10 x 11ml
    Total-MEK1/2 antibody 1 x 9µl 5 x 9µl
  • Research areas

  • Function

    Dual specificity protein kinase which acts as an essential component of the MAP kinase signal transduction pathway. Binding of extracellular ligands such as growth factors, cytokines and hormones to their cell-surface receptors activates RAS and this initiates RAF1 activation. RAF1 then further activates the dual-specificity protein kinases MAP2K1/MEK1 and MAP2K2/MEK2. Both MAP2K1/MEK1 and MAP2K2/MEK2 function specifically in the MAPK/ERK cascade, and catalyze the concomitant phosphorylation of a threonine and a tyrosine residue in a Thr-Glu-Tyr sequence located in the extracellular signal-regulated kinases MAPK3/ERK1 and MAPK1/ERK2, leading to their activation and further transduction of the signal within the MAPK/ERK cascade. Depending on the cellular context, this pathway mediates diverse biological functions such as cell growth, adhesion, survival and differentiation, predominantly through the regulation of transcription, metabolism and cytoskeletal rearrangements. One target of the MAPK/ERK cascade is peroxisome proliferator-activated receptor gamma (PPARG), a nuclear receptor that promotes differentiation and apoptosis. MAP2K1/MEK1 has been shown to export PPARG from the nucleus. The MAPK/ERK cascade is also involved in the regulation of endosomal dynamics, including lysosome processing and endosome cycling through the perinuclear recycling compartment (PNRC), as well as in the fragmentation of the Golgi apparatus during mitosis.
  • Tissue specificity

    Widely expressed, with extremely low levels in brain.
  • Involvement in disease

    Cardiofaciocutaneous syndrome 3
  • Sequence similarities

    Belongs to the protein kinase superfamily. STE Ser/Thr protein kinase family. MAP kinase kinase subfamily.
    Contains 1 protein kinase domain.
  • Domain

    The proline-rich region localized between residues 270 and 307 is important for binding to RAF1 and activation of MAP2K1/MEK1.
  • Post-translational
    modifications

    Phosphorylation at Ser-218 and Ser-222 by MAP kinase kinase kinases (RAF or MEKK1) positively regulates kinase activity. Also phosphorylated at Thr-292 by MAPK1/ERK2 and at Ser-298 by PAK. MAPK1/ERK2 phosphorylation of Thr-292 occurs in response to cellular adhesion and leads to inhibition of Ser-298 phosphorylation by PAK.
    Acetylation by Yersinia yopJ prevents phosphorylation and activation, thus blocking the MAPK signaling pathway.
  • Cellular localization

    Cytoplasm, cytoskeleton, microtubule organizing center, centrosome. Cytoplasm, cytoskeleton, microtubule organizing center, spindle pole body. Cytoplasm. Nucleus. Localizes at centrosomes during prometaphase, midzone during anaphase and midbody during telophase/cytokinesis.
  • Information by UniProt
  • Alternative names

    • AA589381
    • CFC3
    • Dual specificity mitogen-activated protein kinase kinase 1
    • Dual specificity mitogen-activated protein kinase kinase 2
    • EC 2.7.12.2
    • ERK activator kinase 1
    • ERK activator kinase 2
    • FLJ26075
    • MAP kinase kinase 1
    • MAP kinase kinase 2
    • MAP2K1
    • MAP2K2
    • MAPK/ERK kinase 1
    • MAPK/ERK kinase 2
    • MAPKK 1
    • MAPKK1
    • MAPKK2
    • MEK 1
    • MEK1
    • MEKK1
    • Mitogen activated protein kinase kinase 1
    • Mitogen activated protein kinase kinase 2
    • Mitogen-activated protein kinase kinase 2, p45
    • MK2
    • MKK 1
    • MKK 2
    • MKK1
    • MKK2
    • MP2K1_HUMAN
    • PRKMK 1
    • PRKMK 2
    • Prkmk1
    • Prkmk2
    • Protein kinase, mitogen-activated, kinase 1
    • protein kinase, mitogen-activated, kinase 1 (MAP kinase kinase 1)
    • Protein kinase, mitogen-activated, kinase 2
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab207479 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.

Images

  • Measurement of phosphorylated and total MEK1/2. NIH/3T3 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 20% FBS for 30 minutes and fixed. Total and phospho-MEK1/2 were each assayed in triplicate using the phospho-MEK1/2 and total MEK1/2 antibodies included in the kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Protocols

References

ab207479 has not yet been referenced specifically in any publications.

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