RecombinantRabMAb

Recombinant Anti-eNOS antibody [EPR23750-3] (ab252439)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR23750-3] to eNOS
  • Suitable for: IHC-P, ICC, Flow Cyt, WB, IP
  • Reacts with: Human

Overview

  • Product name

    Anti-eNOS antibody [EPR23750-3]
    See all eNOS primary antibodies

  • Description

    Rabbit monoclonal [EPR23750-3] to eNOS

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IHC-P, ICC, Flow Cyt, WB, IPmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Recombinant fragment within Human eNOS aa 700-1200. The exact sequence is proprietary.
    Database link: P29474

  • Positive control

    • WB: EA.hy926 and HUVEC whole cell lysates. IHC-P: Human lung carcinoma, kidney, spleen and placenta tissue. ICC: HUVEC cells. Flow cyt: HeLa cells. IP: EA.hy926 whole cell lysate.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

Properties

Applications

Our Abpromise guarantee covers the use of ab252439 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.
ICC 1/100.
Flow Cyt 1/600.
WB 1/1000. Detects a band of approximately 140 kDa (predicted molecular weight: 133 kDa).
IP 1/30.

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

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (ab252439)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (ab252439)

      Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling eNOS with ab252439 at 1/500 (1.198 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on endothelial cells in human spleen. The section was incubated with ab252439 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

    • Immunocytochemistry/ Immunofluorescence - Anti-eNOS antibody [EPR23750-3] (ab252439)
      Immunocytochemistry/ Immunofluorescence - Anti-eNOS antibody [EPR23750-3] (ab252439)

      Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HUVEC cells labelling eNOS with ab252439 at 1/100 (5.99 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HUVEC cells. Negative control: HeLa (PMID: 19559671). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

    • Flow Cytometry - Anti-eNOS antibody [EPR23750-3] (ab252439)
      Flow Cytometry - Anti-eNOS antibody [EPR23750-3] (ab252439)

      Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human cervix adenocarcinoma epithelial cell, left) /HUVEC (human umbilical vein endothelial cell, right) cells labelling eNOS with ab252439 at 1/50 dilution (1ug)(Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

      A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

      Negative control: Hela (PMID: 19559671).

    • Immunoprecipitation - Anti-eNOS antibody [EPR23750-3] (ab252439)
      Immunoprecipitation - Anti-eNOS antibody [EPR23750-3] (ab252439)

      eNOS was immunoprecipitated from 0.35 mg EA.hy926 (human somatic cell hybrid endothelial) whole cell lysate with ab252439 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab252439 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

      Lane 1: EA.hy926 (human somatic cell hybrid endothelial) whole cell lysate 10 ug

      Lane 2: ab252439 IP in EA.hy926 (human somatic cell hybrid endothelial) whole cell lysate

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252439 in EA.hy926 (human somatic cell hybrid endothelial) whole cell lysate

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

      Exposure time: 6 seconds.

      IP lysates were unboiled.

    • Western blot - Anti-eNOS antibody [EPR23750-3] (ab252439)
      Western blot - Anti-eNOS antibody [EPR23750-3] (ab252439)
      Anti-eNOS antibody [EPR23750-3] (ab252439) at 1/1000 dilution + HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

      Secondary
      Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

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



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

      HUVEC whole cell lysate in lane 1 is made freshly and used in WB test immediately to minimize protein degradation.

      Exposure time: 6 seconds.

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (ab252439)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (ab252439)

      Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue labeling eNOS with abab252439 at 1/500 (1.198 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on endothelial cells in human lung carcinoma. The section was incubated with ab252439 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

    • Western blot - Anti-eNOS antibody [EPR23750-3] (ab252439)
      Western blot - Anti-eNOS antibody [EPR23750-3] (ab252439)
      All lanes : Anti-eNOS antibody [EPR23750-3] (ab252439) at 1/1000 dilution

      Lane 1 : EA.hy926 (human somatic cell hybrid endothelial) whole cell lysate
      Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

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



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

      Negative control: HeLa (PMID:19559671).

      Lysates were made freshly and used in WB test immediately to minimize protein degradation. Lysates were unboiled.

      Exposure time: 6 seconds.

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (ab252439)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (ab252439)

      Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling eNOS with ab252439 at 1/500 (1.198 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining in human placenta. The section was incubated with ab252439 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (ab252439)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody [EPR23750-3] (ab252439)

      Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling eNOS with ab252439 at 1/500 (1.198 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on endothelial cells in human kidney. The section was incubated with ab252439 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

    • Anti-eNOS antibody [EPR23750-3] (ab252439)
      Anti-eNOS antibody [EPR23750-3] (ab252439)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    References (0)

    Publishing research using ab252439? Please let us know so that we can cite the reference in this datasheet.

    ab252439 has not yet been referenced specifically in any publications.

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