Anti-eNOS antibody (ab5589)

Reviews (8)Q&A (26)References (49)

Overview

  • Product name
    Anti-eNOS antibody
    See all eNOS primary antibodies
  • Description
    Rabbit polyclonal to eNOS
  • Host species
    Rabbit
  • Specificity
    This antibody does not detect human inducible NOS or rat brain nNOS. ab5589 has been successfully used in Western blot and immunohistochemistry (paraffin and frozen) procedures. By Western blot, this antibody detects an ~140 kDa protein representing eNOS from human endothelial cells. Immunohistochemical staining of eNOS in Human vascular endothelial cells with ab5589 results in prominent staining of the epithelial cells.
  • Tested applications
    Suitable for: ICC/IF, IHC-P, IHC-Fr, WBmore details
  • Species reactivity
    Reacts with: Rabbit, Guinea pig, Cow, Dog, Human, Pig
  • Immunogen

    Synthetic peptide corresponding to Human eNOS aa 1179-1194.
    Sequence:

    SLQERQLRGAVPWAFD

    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI
  • Positive control
    • IHC: Human endothelial cells. WB:canine cardiac microsomes and rat or human lung lysate.
  • General notes

    We have received mixed feedback from customers regarding rat specificity; this is why we are temporarily removing Rat from tested species list and no longer guarantee it. Our laboratory is currently testing new lots so please contact our scientific support team (technical@abcam.com) for more information.

Properties

Applications

Our Abpromise guarantee covers the use of ab5589 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
ICC/IF Use at an assay dependent concentration. PubMed: 16797727
IHC-P 1/100. Antigen retrieval is not essential but may optimise staining.
IHC-Fr 1/100.
WB 1/1000. Predicted molecular weight: 133 kDa.

By Western blot ab5589 also detects eNOS in canine cardiac microsomes.
5% BSA in TBST is recommended as blocking buffer.

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
    see all
  • 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 (ab5589)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody (ab5589)

    IHC image of eNOS staining in human placenta formalin fixed paraffin embedded tissue section*, 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 ab5589, 1/100, 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

  • Western blot - Anti-eNOS antibody (ab5589)
    Western blot - Anti-eNOS antibody (ab5589)
    Lanes 1-4 : Anti-eNOS antibody (ab5589) at 1/200 dilution
    Lanes 5-8 : Anti-eNOS antibody (ab5589) at 1/600 dilution
    Lanes 9-12 : Anti-eNOS antibody (ab5589) at 1/1800 dilution
    Lanes 13-16 : Anti-eNOS antibody (ab5589) at 1/5400 dilution

    Lanes 1 & 5 & 9 & 13 : Huvec
    Lanes 2 & 6 & 10 & 14 : Raw264.7
    Lanes 3 & 7 & 11 & 15 : Mouse Placenta
    Lanes 4 & 8 & 12 & 16 : Rat Placenta

    Lysates/proteins at 10 µg per lane.

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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody (ab5589)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-eNOS antibody (ab5589)

    Immunolocalization of eNOS in human lung tissue using ab5589.

References

This product has been referenced in:
  • Voss NCS  et al. Enhanced nitric oxide signaling amplifies vasorelaxation of human colon cancer feed arteries. Am J Physiol Heart Circ Physiol 316:H245-H254 (2019). Read more (PubMed: 30444664) »
  • Guerby P  et al. High glutathionylation of placental endothelial nitric oxide synthase in preeclampsia. Redox Biol 22:101126 (2019). Read more (PubMed: 30738311) »
See all 49 Publications for this product

Publishing research using ab5589? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 34 Abreviews or Q&A

Question

We want to perform western blotting on HUVECs (Human Umbilical Endothelial Vein Cells) and A7R5 cells lines (rat aortic smooth muscle cell), both of the cell lines contain eNOS. As we found various sources of anti-eNOS antibody for WB on Abcam webpage, such as ab66127, ab5589 and ab51086, we are wondering which one could be suitable for our needs?

Read More
Answer

Any of the following antibodies would be suitable as they are guaranteed to work in human, rat and in WB:

Anti-eNOS antibody (ab66127)
Anti-eNOS antibody (ab5589)
Anti-eNOS antibody (ab51086)
Anti-eNOS antibody [NOS-E1] (ab50010)

Out of these antibodies, I would suggest using ab5589 as we have had one customer who used this in western blot and obtained good results. In addition, this antibody has also been extensively used in WB in several publications.

Read More

Immunohistochemistry (Frozen sections) abreview for Anti-eNOS antibody

 Good
Abreviews
Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Sheep Tissue sections (carotid artery)
Permeabilization
Yes - PBS-tween
Specification
carotid artery
Blocking step
Serum as blocking agent for 10 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

Submitted Feb 20 2019

Western blot abreview for Anti-eNOS antibody

 Good
Abreviews
abreview image
Application
Western blot
Sample
Cow Cell lysate - whole cell (Aortic Endothelial Cells)
Gel Running Conditions
Reduced Denaturing (4-15%)
Loading amount
10 µg
Specification
Aortic Endothelial Cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Read More

Abcam user community

Verified customer

Submitted Oct 19 2017

Western blot abreview for Anti-eNOS antibody

 Average
Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
abreview image
Application
Western blot
Sample
Pig Cell lysate - whole cell (endothelial cell)
Gel Running Conditions
Non-reduced Denaturing (8% gel)
Loading amount
30 µg
Specification
endothelial cell
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Read More

Abcam user community

Verified customer

Submitted Jul 24 2015

Western blot abreview for Anti-eNOS antibody

 Excellent
Abreviews
Application
Western blot
Loading amount
15 µg
Gel Running Conditions
Non-reduced Denaturing (7.5%)
Sample
Human Cell lysate - whole cell (endothelial cells)
Specification
endothelial cells
Treatment
vegf stimulated
Blocking step
Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Read More

Vidhya Ramaswamy

Verified customer

Submitted Sep 30 2014

Immunohistochemistry (PFA perfusion fixed frozen sections) abreview for Anti-eNOS antibody

 Excellent
Abreviews
abreview image
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Blocking step
BSA as blocking agent for 45 minute(s) · Concentration: 1% · Temperature: 25°C
Antigen retrieval step
None
Sample
Rat Tissue sections (Mesenteric venule)
Specification
Mesenteric venule
Permeabilization
Yes - Triton X-100
Fixative
Paraformaldehyde
Read More

Sulei Xu

Verified customer

Submitted Nov 13 2013

Western blot abreview for Anti-eNOS antibody

 Good
Abreviews
abreview image
Application
Western blot
Sample
Mouse Cell lysate - whole cell (lung endothelial cells)
Gel Running Conditions
Reduced Denaturing (gradient gel)
Loading amount
10 µg
Specification
lung endothelial cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Read More

Abcam user community

Verified customer

Submitted Apr 12 2013

Question

Band around 25-30 kDa in Western blot instead of around 130-140 kDa as expected.

Read More
Answer

Thank you very much for your call today and for letting us know about the trouble with this antibody.

As we discussed, I'm sending a free of charge vial of ab66217 which should arrive tomorrow.

Please keep me updated about the results after trying this antibody, and let me know if there is anything else that we can do for you. I'll be happy to help.

Read More

Question

Looking for antibodies to use in ICC with human cells.

Read More
Answer

Thank you so much for your calls today and for your questions.

Please let me know if you would like to try any of the antibodies in ICC that haven't already been tested, and I will be happy to set up the testing discount program for you. If you have any questions or need anything else, please don't hesitate to ask.

Have a great day!

Read More

Question

phone call: searching antibody for eNOS and phospho eNOS on rat and mouse tissue, on IHC-P and WB.

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Answer

A while ago we spoke in regards to searching for the best antibodies to use for detection of eNOS and phospho eNOS on mouse and rat tissue, on paraffin sections (IHC-P)as well as in Western blot.
..

We have the ab66127 or the ab5589 against total eNOS which have been tested in mouse and rat, as well as in IHC-P and Western blot. These antibodies are therefore guaranteed by us for this use.
https://www.abcam.com/index.html?datasheet=66127 (or use the following: https://www.abcam.com/index.html?datasheet=66127).
https://www.abcam.com/index.html?datasheet=5589 (or use the following: https://www.abcam.com/index.html?datasheet=5589).

As an antibody against phsopho eNOS, we do have the ab75639 which recognizes the S1177 on eNOS. This antibody is again tested and guaranteed in mouse, rat and IHC-P and Western blot.
https://www.abcam.com/index.html?datasheet=75639 (or use the following: https://www.abcam.com/index.html?datasheet=75639).

I have followed up with the laboratory in regards to the protocols used for the ab75639. For the Western blot, the cell lysates have been prepared with protease and phosphatase inhibitors. Before loading, the lysates have been denatured and reduced. Importantly, the membranes were blocked with 5% BSA in TBS-T. The IHC-P has been performed with heat induced antigen retrieval in Citrate buffer pH 6.

I hope this information is still useful. Please do not hesitate to let me know if you have any question or if you require any other information from our side in regards to these or other antibodies.

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1-10 of 34 Abreviews or Q&A

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