Key features and details
- Sample type: Adherent cells, Suspension cells
- Detection method: Colorimetric
- Assay type: Cell-based (quantitative)
- Reacts with: Mouse, Rat, Human
Product namePhospho-MEK1/2 (S217/S221) + Total In-Cell ELISA Kit
See all MEK1 + MEK2 kits
Sample typeAdherent cells, Suspension cells
Assay typeCell-based (quantitative)
Assay durationMultiple steps standard assay
Species reactivityReacts with: Mouse, Rat, Human
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.
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.
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Storage instructionsPlease 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
FunctionDual 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 specificityWidely expressed, with extremely low levels in brain.
Involvement in diseaseCardiofaciocutaneous syndrome 3
Sequence similaritiesBelongs to the protein kinase superfamily. STE Ser/Thr protein kinase family. MAP kinase kinase subfamily.
Contains 1 protein kinase domain.
DomainThe proline-rich region localized between residues 270 and 307 is important for binding to RAF1 and activation of MAP2K1/MEK1.
modificationsPhosphorylation 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 localizationCytoplasm, 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
- Dual specificity mitogen-activated protein kinase kinase 1
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).
ab207479 has not yet been referenced specifically in any publications.