Anti-eNOS antibody [M221] (ab76198)

Mouse monoclonal eNOS antibody [M221]. Validated in WB, ELISA, IHC, Flow Cyt, ICC/IF and tested in Mouse, Rat, Human. Cited in 22 publication(s). Independently reviewed in 11 review(s).

Overview

  • Product name
    Anti-eNOS antibody [M221]
    See all eNOS primary antibodies
  • Description
    Mouse monoclonal [M221] to eNOS
  • Host species
    Mouse
  • Specificity
    ab76198 is not predicted to react with other NOS family members due to low homology.
  • Tested applications
    Suitable for: IHC-P, ICC/IF, Flow Cyt, WB, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Recombinant fragment of mouse eNOS protein that included amino acid residues in the C terminal region.

  • Positive control
    • Human umbilical vein endothelial cells untreated and treated with lambda phosphatase. Mouse placenta lysate. Huvec lysate. IHC-P: FFPE human normal placenta tissue sections.
  • General notes

    This antibody detects eNOS in mouse and rat but at a lower intensity than in human. If you are working in mouse or rat, we would recommend using no more than 1% milk as the blocking agent for optimal signal strength.

Properties

Applications

Our Abpromise guarantee covers the use of ab76198 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 1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF Use at an assay dependent concentration.
Flow Cyt Use 2µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

WB 1/500 - 1/1000. Predicted molecular weight: 133 kDa.
ELISA 1/2000.

Target

  • Function
    Produces nitric oxide (NO) which is implicated in vascular smooth muscle relaxation through a cGMP-mediated signal transduction pathway. NO mediates vascular endothelial growth factor (VEGF)-induced angiogenesis in coronary vessels and promotes blood clotting through the activation of platelets.
    Isoform eNOS13C: Lacks eNOS activity, dominant-negative form that may down-regulate eNOS activity by forming heterodimers with isoform 1.
  • Tissue specificity
    Platelets, placenta, liver and kidney.
  • Involvement in disease
    Variation in NOS3 seem to be associated with susceptibility to coronary spasm.
  • Sequence similarities
    Belongs to the NOS family.
    Contains 1 FAD-binding FR-type domain.
    Contains 1 flavodoxin-like domain.
  • Post-translational
    modifications
    Phosphorylation by AMPK at Ser-1177 in the presence of Ca(2+)-calmodulin (CaM) activates activity. In absence of Ca(2+)-calmodulin, AMPK also phosphorylates Thr-495, resulting in inhibition of activity (By similarity). Phosphorylation of Ser-114 by CDK5 reduces activity.
  • Cellular localization
    Cell membrane. Membrane, caveola. Cytoplasm, cytoskeleton. Golgi apparatus. Specifically associates with actin cytoskeleton in the G2 phase of the cell cycle and which is favored by interaction with NOSIP and results in a reduced enzymatic activity.
  • Information by UniProt
  • Database links
  • Alternative names
    • cNOS antibody
    • Constitutive NOS antibody
    • EC NOS antibody
    • EC-NOS antibody
    • ecNOS antibody
    • Endothelial nitric oxidase synthase antibody
    • Endothelial nitric oxide synthase antibody
    • Endothelial nitric oxide synthase 3 antibody
    • Endothelial NOS antibody
    • eNOS antibody
    • Nitric oxide synthase 3 (endothelial cell) antibody
    • Nitric oxide synthase 3 antibody
    • Nitric oxide synthase 3 endothelial cell antibody
    • Nitric oxide synthase endothelial antibody
    • Nitric oxide synthase, endothelial antibody
    • NOS 3 antibody
    • NOS III antibody
    • NOS type III antibody
    • NOS3 antibody
    • NOS3_HUMAN antibody
    • NOSIII antibody
    see all

Images

  • All lanes : Anti-eNOS antibody [M221] (ab76198) at 1/500 dilution

    Lane 1 : Huvec cell lysates
    Lane 2 : Mouse placenta lysates

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat anti Mouse IR680 at 1/10000 dilution

    Predicted band size: 133 kDa



    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab76198 overnight at 4°C. Antibody binding was detected using Goat anti Mouse IR680 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  • IHC image of eNOS staining in human normal placenta*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab76198, 1/1000 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • ab76198 staining eNOS in human umbilical vein endothelial cells. Cells with fixed with paraformaldehyde.

     

  • Overlay histogram showing HEK293 cells stained with ab76198 (red line). The cells were fixed with 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 (ab76198, 2µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

  • All lanes : Anti-eNOS antibody [M221] (ab76198) at 1/1000 dilution

    Lane 1 : human umbilical vein endothelial
    cells, untreated
    Lane 2 : human umbilical vein endothelial
    cells, treated with lambda phosphatase

    Predicted band size: 133 kDa
    Observed band size: 140 kDa
    why is the actual band size different from the predicted?

References

This product has been referenced in:
  • Vilà-González M  et al. Enhanced Function of Induced Pluripotent Stem Cell-Derived Endothelial Cells Through ESM1 Signaling. Stem Cells 37:226-239 (2019). Read more (PubMed: 30372556) »
  • Zhang S  et al. Matrine attenuates high-fat diet-induced in vivo and ox-LDL-induced in vitro vascular injury by regulating the PKCa/eNOS and PI3K/Akt/eNOS pathways. J Cell Mol Med 23:2731-2743 (2019). Read more (PubMed: 30770623) »
See all 40 Publications for this product

Customer reviews and Q&As

Answer


Our polyclonal antibodies are generally provided in greater total micrograms than our monoclonal antibodies even though the cost is comparable. The reasons for this are outlined in the comparison list through the link below, explaining that monoclonal antibodies are more expensive to produce compared to polyclonal antibodies:
https://www.abcam.com/index.html?pageconfig=resource&rid=11269&pid=11287

As ab76198 is a mouse monoclonal antibody, it is provided at a slightly lower total amount compared to the polyclonal antibodies. However, ab76198 will still last a comparable amount of time for western blot (1/1000 recommended dilution) and ELISA (1/2000 recommended dilution), and is sufficient for 6 flow cytometry experiments of 10^6 cells each.

Read More

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

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