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Phospho-MEK1 (Thr292) Polyclonal Antibody
Antibody target was verified by Cell Treatment to ensure the antibody binds to the antigen stated.
Catalog # 44-458G
(USD) 309.00, 10 blots
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Immunofluorescence analysis of Phospho-MEK1 pThr292 Antibody was done on 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-MEK1 pThr292 Antibody (Product # 44-458G) at 1:250 dilution in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa flour 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing nuclear localization. Panel e is a no primary antibody control. The images were captured at 40X magnification.
Immunohistochemistry analysis of MEK1 (pT292) showing staining in the cytoplasm of paraffin-embedded human kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a MEK1 (pT292) polyclonal antibody (Product # 44-458G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
Peptide Competition Extracts of NIH3T3 cells untreated (lane 1) or treated with 50 ng/mL PDGF for 15 minutes (lanes 2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 4% BSA-TBST buffer for one hour at room temperature, then incubated with the MEK1 [pT292] antibody (Product # 44-458G) in a 1% BSA-TBST buffer for two hours at room temperature, following prior incubation with: no peptide (1, 2), a generic phosphothreonine-containing peptide (3), the non-phosphopeptide corresponding to the phosphopeptide immunogen (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to MEK1 [pT292] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of MEK1 [pT292] phosphorylation by the addition of PDGF to this cell system. In addition, this antibody did not recognize a recombinant MEK1 T292A mutant protein (kindly provided by Dr. Natalie Ahn, University of Colorado), further demonstrating its specificity for MEK1 [pT292] (data not shown).
Western blot analysis of MEK1 (pT292) was performed by loading 20 µg of PC-12 (lane1) and NIH\u0003T3 (lane2) cell lysate using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® 2 Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. MEK1 (pT292) was detected at ~ 43 kDa using MEK1 (pT292) Rabbit Polyclonal Antibody (Product # 44-244G) at 1:1000 dilution in 5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
Altered expression of proteins upon cell treatment demonstrates antibody specificity. Western blot of Phospho-MEK1 (Thr292) using MEK1 [pT292] Rabbit Polyclonal Antibody (Product # 44-458G), shows increase in expression Phospho-MEK1 (Thr292)in NIH3T3 cells treated with PDGF.
Immunohistochemistry (Paraffin) (IHC (P))
Western Blot (WB)
Host / Isotype
The antiserum was produced against a chemically synthesized phosphopeptide derived from a region of human MEK1 that contains threonine 292. The sequence is conserved in many species including mouse, rat, chimp, hamster and rabbit.
Antigen affinity chromatography
Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/ml BSA
0.05% sodium azide
MAP2K1 is a dual specificity mitogen-activated protein kinase (MAPK) kinase. It is an essential component of the MAP kinase pathway and stimulates the enzymatic activity of MAP kinases in response to various signals. Growth factors, cytokines, and proto-oncogenes transduce their signals through the activation of RAS, which leads to activation of the serine/threonine kinase RAF, and then to activation of MAPK kinase. MAP2K1 is activated by the Raf family member p74raf-1, through phosphorylation of serine residues at positions 217 and 221 in the activation loop.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
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Protein Aliases: Dual specificity mitogen-activated protein kinase kinase 1; EC 184.108.40.206; ERK activator kinase 1; kinase MEK1; MAP kinase kinase 1; MAP kinase/Erk kinase 1; MAP2K1; MAPK/ERK kinase 1; MAPKK 1; MAPKK1; MEK 1; mitogen activated protein kinase kinase 1; mitogen-activated protein kinase kinase 1; MP2K1; PRKMK1; protein kinase, mitogen activated, kinase 1, p45; protein kinase, mitogen-activated, kinase 1 (MAP kinase kinase 1)
Gene Aliases: CFC3; MAP2K1; MAPKK1; MEK1; MEKK1; MKK1; PRKMK1
UniProt ID: (Human) Q02750, (Mouse) P31938, (Rat) Q01986
Entrez Gene ID: (Human) 5604, (Mouse) 26395, (Rat) 170851
Suggested Secondary Antibodies
Material safety data sheets (MSDS)