Overview

  • Product name
    Anti-ERK5 antibody [EP791Y]
    See all ERK5 primary antibodies
  • Description
    Rabbit monoclonal [EP791Y] to ERK5
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, Flow Cyt, IP, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human ERK5 aa 800-900 (C terminal). The exact sequence is proprietary.

  • Positive control
    • Hela Cell lysate.
  • 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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab40809 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
WB 1/10000. Detects a band of approximately 115 kDa (predicted molecular weight: 89 kDa).

For unpurified use at 1/1000 - 1/5000 dilution. 

Flow Cyt 1/50.

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

For unpurified use at 1/100 dilution. 

IP 1/30.

For unpurified use at 1/50 dilution.

ICC/IF 1/100.

For unpurified use at 1/250 - 1/500 dilution.

Target

  • Function
    Plays a role in various cellular processes such as proliferation, differentiation and cell survival. The upstream activator of MAPK7 is the MAPK kinase MAP2K5. Upon activation, it translocates to the nucleus and phosphorylates various downstream targets including MEF2C. EGF activates MAPK7 through a Ras-independent and MAP2K5-dependent pathway. May have a role in muscle cell differentiation. May be important for endothelial function and maintenance of blood vessel integrity. MAP2K5 and MAPK7 interact specifically with one another and not with MEK1/ERK1 or MEK2/ERK2 pathways.
  • Tissue specificity
    Expressed in many adult tissues. Abundant in heart, placenta, lung, kidney and skeletal muscle. Not detectable in liver.
  • Sequence similarities
    Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. MAP kinase subfamily.
    Contains 1 protein kinase domain.
  • Domain
    The second proline-rich region may interact with actin targeting the kinase to a specific location in the cell.
    The TXY motif contains the threonine and tyrosine residues whose phosphorylation activates the MAP kinases.
  • Post-translational
    modifications
    Dually phosphorylated on Thr-219 and Tyr-221, which activates the enzyme (By similarity). Autophosphorylated in vitro on threonine and tyrosine residues when the C-terminal part of the kinase, which could have a regulatory role, is absent.
  • Cellular localization
    Cytoplasm. Nucleus. Translocates to the nucleus upon activation.
  • Information by UniProt
  • Database links
  • Alternative names
    • Big MAP kinase 1 antibody
    • BMK 1 antibody
    • BMK 1 kinase antibody
    • BMK-1 antibody
    • BMK1 antibody
    • BMK1 Kinase antibody
    • EC 2.7.11.24 antibody
    • ERK 4 antibody
    • ERK 5 antibody
    • ERK-5 antibody
    • ERK4 antibody
    • ERK5 antibody
    • Extracellular signal regulated kinase 5 antibody
    • Extracellular signal-regulated kinase 5 antibody
    • MAP kinase 7 antibody
    • MAPK 7 antibody
    • MAPK7 antibody
    • Mitogen activated protein kinase 7 antibody
    • Mitogen-activated protein kinase 7 antibody
    • MK07_HUMAN antibody
    • OTTHUMP00000065906 antibody
    • OTTHUMP00000065907 antibody
    • PRKM 7 antibody
    • PRKM7 antibody
    • PROTEIN KINASE, MITOGEN-ACTIVATED, 7 antibody
    see all

Images

  • Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
    Lane 2: MAPK7 (ERK5) knockout HAP1 whole cell lysate (20 µg)
    Lane 3: HeLa whole cell lysate (20 µg)

    Lanes 1 - 3: Merged signal (red and green). Green - ab40809 observed at 88 kDa. Red - loading control, ab9484, observed at 37 kDa.

    Unpurified ab40809 was shown to specifically react with ERK5 in wild-type HAP1 cells as signal was lost in MAPK7 (ERK5) knockout cells. Wild-type and MAPK7 (ERK5) knockout samples were subjected to SDS-PAGE. Unpurified ab40809 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ERK5 with Purified ab40809 at 1:100 (4.8 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 μg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ERK5 with purified ab40809 at 1:50 dilution (10 ug/ml) (red). Cells were fixed with 80% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

  • All lanes : Anti-ERK5 antibody [EP791Y] (ab40809) at 1/2000 dilution (Purified)

    Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates with 5% NFDM/TBST
    Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates with 5% NFDM/TBST
    Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates with 5% NFDM/TBST

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 89 kDa
    Observed band size: 115 kDa
    why is the actual band size different from the predicted?

  • Anti-ERK5 antibody [EP791Y] (ab40809) at 1/5000 dilution (unpurified) + Hela cell lysate at 10 µg

    Predicted band size: 89 kDa
    Observed band size: 115 kDa why is the actual band size different from the predicted?



    The predicted weight of 89 kDa is for the precursor version of human ERK5 protein. However, ab40809 detects endogenous levels of total Erk5 protein which appears around 115 kDa in SDS PAGE
  • Overlay histogram showing A549 cells stained with unpurified ab40809 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab40809, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

  • ab40809 (purified) at 1:30 dilution (2ug) immunoprecipitating ERK5 in HeLa whole cell lysate.
    Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
    Lane 2 (+): ab40809 & HeLa whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab40809 in HeLa whole cell lysate
    For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1:1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.

References

This product has been referenced in:
  • Li L  et al. Thymic Stromal Lymphopoietin Promotes Fibrosis and Activates Mitogen-Activated Protein Kinases in MRC-5 Cells. Med Sci Monit 22:2357-62 (2016). WB ; Human . Read more (PubMed: 27385084) »
  • Xiong Y  et al. Phosphorylation of BMK1 induces prostatic carcinoma cell proliferation by promoting entry into the S phase of the cell cycle. Oncol Lett 11:99-104 (2016). Read more (PubMed: 26870175) »
See all 5 Publications for this product

Customer reviews and Q&As

Answer

Following up my previous message, the lab confirmed that ab40809 was negative for IHC on formalin -fixed tissue. However, it was positive for ICC and flow cytometry on PFA-fixed samples. These application use relatively short fixation times compared to IHC samples. It is possible that the relatively shortcross-linking step for ChIP will also allow a positive result, but we have no data.

Read More

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