Validated using a knockout cell line
Recombinant
RabMAb

Anti-MEK3 antibody [EPR17345-104] (ab195037)

Overview

  • Product name
    Anti-MEK3 antibody [EPR17345-104]
    See all MEK3 primary antibodies
  • Description
    Rabbit monoclonal [EPR17345-104] to MEK3
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, WB, ICC/IF, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Recombinant fragment within Human MEK3 aa 150 to the C-terminus. The exact sequence is proprietary.
    Database link: P46734

  • Positive control
    • WB: HepG2, HeLa and MCF7 whole cell lysates; Human skeletal muscle, mouse spleen and rat spleen lysates. IHC-P: Human skeletal muscle, Human lung adenocarcinoma, mouse skeletal muscle and rat skeletal muscle tissues. ICC/IF: NIH/3T3 cells. IP: HeLa whole cell extract.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab195037 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
IHC-P 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/5000. Detects a band of approximately 39, 34 kDa (predicted molecular weight: 39 kDa).
ICC/IF 1/250.
IP 1/90.
Flow Cyt 1/1000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

 

Target

  • Function
    Dual specificity kinase. Is activated by cytokines and environmental stress in vivo. Catalyzes the concomitant phosphorylation of a threonine and a tyrosine residue in the MAP kinase p38.
  • Tissue specificity
    Abundant expression is seen in the skeletal muscle. It is also widely expressed in other tissues.
  • Involvement in disease
    Note=Defects in MAP2K3 may be involved in colon cancer.
  • Sequence similarities
    Belongs to the protein kinase superfamily. STE Ser/Thr protein kinase family. MAP kinase kinase subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Autophosphorylated.
    Phosphorylation on Ser-218 and Thr-222 by MAP kinase kinase kinases regulates positively the kinase activity.
    Yersinia yopJ may acetylate Ser/Thr residues, preventing phosphorylation and activation, thus blocking the MAPK signaling pathway.
  • Information by UniProt
  • Database links
  • Alternative names
    • AW212142 antibody
    • dual specificity mitogen activated protein kinase kinase 3 antibody
    • Dual specificity mitogen-activated protein kinase kinase 3 antibody
    • MAP kinase kinase 3 antibody
    • map2k3 antibody
    • MAPK ERK kinase 3 antibody
    • MAPK/ERK kinase 3 antibody
    • MAPKK 3 antibody
    • MAPKK3 antibody
    • MEK 3 antibody
    • MEK3 antibody
    • Mitogen activated protein kinase kinase 3 antibody
    • MKK 3 antibody
    • MKK3 antibody
    • mMKK3b antibody
    • MP2K3_HUMAN antibody
    • PRKMK 3 antibody
    • PRKMK3 antibody
    • protein kinase, mitogen-activated, kinase 3 antibody
    • SAPK kinase 2 antibody
    • SAPKK 2 antibody
    • SAPKK2 antibody
    • Stress activated protein kinase kinase 2 antibody
    see all

Images

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: MEK3 knockout HAP1 cell lysate (20 µg)
    Lane 3: HeLa cell lysate (20 µg)
    Lane 4: Jurkat cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab195037 observed at 40 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab195037 was shown to specifically react with MEK3 when MEK3 knockout samples were used. Wild-type and MEK3 knockout samples were subjected to SDS-PAGE. ab195037 and ab8245 (loading control to GAPDH) were both diluted to 1/5000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW)  preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling MEK3 with ab195037 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic and nucleus staining on Human skeletal muscle is observed. Counter stained with Hematoxylin.

    Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

     

    1:Ben-Levy R,et.al.(1998) Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr Biol, 8(19):1049-1057.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embyro fibroblast cells) cells labeling MEK3 with ab195037 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing both nuclear and cytoplasmic staining on NIH/3T3 cell line.  The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab195037 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
    -ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

  • Anti-MEK3 antibody [EPR17345-104] (ab195037) at 1/50000 dilution + HepG2 (Human liver hepatocellular carcinoma) whole cell lysates at 10 µg

    Secondary
    Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

    Predicted band size: 39 kDa
    Observed band size: 34,39 kDa (why is the actual band size different from the predicted?)



    Blocking/Dilution buffer: 5% NFDM/TBST.

     

    Based on the sequence analysis, ab195037 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms.

  • Flow Cytometry analysis of NIH/3T3 (mouse embryo) labelling MEK3 with purified ab195037 at 1/1000 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Alexa Fluor® 488 goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

     

  • All lanes : Anti-MEK3 antibody [EPR17345-104] (ab195037) at 1/10000 dilution

    Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
    Lane 2 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysates

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

    Predicted band size: 39 kDa
    Observed band size: 34,39 kDa (why is the actual band size different from the predicted?)



    Blocking/Dilution buffer: 5% NFDM/TBST.

     

    Based on the sequence analysis, ab195037 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms.

  • Immunohistochemical analysis of paraffin-embedded Human lung adenocarcinoma tissue labeling MEK3 with ab195037 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic and nuclear staining on cancer cells of Human lung adenocarcinoma is observed. Counter stained with Hematoxylin.

    Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • Anti-MEK3 antibody [EPR17345-104] (ab195037) at 1/5000 dilution + Human skeletal muscle lysates at 10 µg

    Secondary
    Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

    Predicted band size: 39 kDa
    Observed band size: 34,39 kDa (why is the actual band size different from the predicted?)



    Blocking/Dilution buffer: 5% NFDM/TBST.

     

    Based on the sequence analysis, ab195037 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms.

  • All lanes : Anti-MEK3 antibody [EPR17345-104] (ab195037) at 1/10000 dilution

    Lane 1 : Mouse spleen lysates
    Lane 2 : Rat spleen lysates

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

    Predicted band size: 39 kDa



    Blocking/Dilution buffer: 5% NFDM/TBST.

     

    Based on the sequence analysis, ab195037 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms.

  • Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling MEK3 with ab195037 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic and nuclear staining on mouse skeletal muscle is observed. Counter stained with Hematoxylin.

    Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • Immunohistochemical analysis of paraffin-embedded Rat skeletal muscle tissue labeling MEK3 with ab195037 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic and nuclear staining on rat skeletal muscle is observed. Counter stained with Hematoxylin.

    Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  • MEK3 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab195037 at 1/90 dilution. Western blot was performed from the immunoprecipitate using ab195037 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1: HeLa whole cell extract. Lane 2: PBS instead of HeLa whole cell extract.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

     

    Based on the sequence analysis, ab195037 recognizes 3 isoforms of MEK3 ranging from 36kDa to 40KDa. The multi-bands should be MEK3 isoforms.

References

ab195037 has not yet been referenced specifically in any publications.

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