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Nitric oxide synthase (endothelial) polyclonal antibody

 
ALX-210-505-R100 100 µl 416.00 USD
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Replaces Prod. #: ALX-210-505/1

Product Specification

Alternative Name:eNOS, NOS III
 
Host:Rabbit
 
Immunogen:Synthetic peptide corresponding to aa 598-612 (P598YNSSPRPEQHKSYK612C) of bovine endothelial nitric oxide synthase (eNOS; NOS III).
 
UniProt ID:P29473
 
Species reactivity:Human, Mouse, Rat
Dog
 
Crossreactivity:Does not cross-react with inducible NOS (iNOS; NOS II) or neuronal NOS (nNOS; NOS I).
 
Applications:ICC, WB
 
Recommended Dilutions/Conditions:Immunocytochemistry (1:250)
Western Blot (1:1,000)
Suggested dilutions/conditions may not be available for all applications.
Optimal conditions must be determined individually for each application.
 
Application Notes:Immunohistochemical staining of eNOS (NOS III) in mouse cardiac tissue with Prod. No. ALX-210-505 results in the staining of the endothelium.
Detects a band of ~140kDa by Western blot.
 
Formulation:Liquid. Neat serum diluted in PBS containing 0.05% sodium azide.
 
Handling:Avoid freeze/thaw cycles.
 
Shipping:Shipped on Blue Ice
 
Long Term Storage:-20°C
 
Protocol:Immunocytochemistry of NOS
  1. Grow cells to near confluence on culture-well slides in RPMI 1640 medium containing 10% bovine calf serum, 2mM glutamine and penicillin/streptomycin.
  2. For study of iNOS, treat cells for 16 hours with fresh medium containing either: no addition (control), interferon-γ (50ng/ml), LPS (30µg/ml) or IFN and LPS.
  3. Wash cells 3 x 5 minutes in PBS.
  4. Permeabilize with 2% saponin for 10 minutes at room temperature.
  5. Fix in 4% formalin for 10 minutes at room temperature.
  6. Wash 3 x 5 minutes in PBS.
  7. Incubate for 20 minutes at room temperature with antibody to eNOS (Prod. No. ALX-210-505/1) diluted with PBS at suggested starting dilution.
  8. Wash 3 x 5 minutes in PBS.
  9. Incubate with biotinylated secondary antibody to rabbit, then with an avidin-biotin complex conjugated to alkaline phosphatase.
  10. React to effect with Fast Red.

Western blot of NOS
  1. For the study of iNOS, prepare cytosolic extracts from cells that are not treated and do not express iNOS activity (control) or cells induced to synthesize NO by pretreatment for 16 hours with a combination pf LPS (30µg/ml) and interferon-γ (50ng/ml).
  2. Run cell extracts on SDS-PAGE using a 4-12% TRIS-glycine gel. Transfer to blotting membrane.
  3. Block membrane in 5%dried milk in PBS containing 1% Tween-20 (PBS-T).
  4. Wash 3 x 5 minutes in PBS.
  5. Incubate for 1 hour at room temperature with antibody to NOS diluted in PBS.
  6. Wash 3 x 5 minutes in PBS.
  7. Incubate for 1 hour with secondary antibody to rabbit coupled to biotin, then with streptavidin conjugated to alkaline phosphatase.
  8. Wash 3 x 5 minutes in PBS.
  9. React to effect with BCIP or Lumi-Phos 530

Note: The procedures listed above are intended only as a guide. Optimal conditions must be determined individually for each application.  
 

Product Literature References

Pulmonary Macrophages Attenuate Hypoxic Pulmonary Vasoconstriction via β3AR/iNOS Pathway in Rats Exposed to Chronic Intermittent Hypoxia: H. Nagai, et al.; PLoS One 10, e0131923 (2015), Application(s): Immunocytochemistry, Western Blot, Abstract; Full Text
Decreased expression of myocardial eNOS and caveolin in dogs with hypertrophic cardiomyopathy: A. Piech, et al.; Am. J. Physiol. Heart Circ. Physiol. 282, H219 (2002), Abstract;
Granulated metrial gland cells contain nitric oxide synthases during pregnancy in the rat: S.M. Sladek, et al.; Placenta 19, 55 (1998), Abstract;
Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts: L.M. Akyurek, et al.; Am. J. Pathol. 149, 1981 (1996), Abstract;
Reduced gene expression of vascular endothelial NO synthase and cyclooxygenase-1 in heart failure: C.J. Smith, et al.; Circ. Res. 78, 58 (1996), Abstract; Full Text
Physiological dilation of uteroplacental arteries in the guinea pig depends on nitric oxide synthase activity of extravillous trophoblast: A. Nanaev, et al.; Cell Tissue Res. 282, 407 (1995), Abstract;

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