Product Specification
| Alternative Name: | iNOS, NOS II |
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| Host: | Rabbit |
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| Immunogen: | Synthetic peptide corresponding to aa 1131-1144 (CK1131KGSALEEPKATRL1144) of mouse inducible nitric oxide synthase (iNOS; NOS II). |
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| UniProt ID: | P29477 |
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| Gene/Protein Identifier: | NM_010927 (RefSeq) |
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| Species reactivity: | Mouse
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| Crossreactivity: | Does not cross-react with eNOS (NOS III) and nNOS (NOS I). |
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| Applications: | IF, IHC (PS), WB
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| Recommended Dilutions/Conditions: | Immunofluorescence (1:100-1:1,000)
Immunohistochemistry (paraffin sections, 1:200)
Immunohistochemistry (frozen sections, 1:500)
Western Blot (1:2,000)
Suggested dilutions/conditions may not be available for all applications.
Optimal conditions must be determined individually for each application. |
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| Application Notes: | Detects a band of ~130kDa by Western blot. |
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| Formulation: | Liquid. Antiserum diluted in PBS containing 0.05% sodium azide. |
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| Handling: | Avoid freeze/thaw cycles. |
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| Shipping: | Shipped on Blue Ice |
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| Long Term Storage: | -20°C |
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| Protocol: | 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
Immunohistochemical Detection of iNOS using PAb to iNOS
1. Heat formalin fixed, paraffin embedded sections in 60°C oven for 35 minutes. Note that samples must first be induced for iNOS expression.
2. De-paraffinize in 100% xylene at 60°C for 35 minutes. Change to fresh xylene 2 x 5 minutes at room temperature.
3. Re-hydrate section in graded ethanol as follows:
- 2 x 5 minutes in 100% ethanol
- 2 x 5 minutes in 95% ethanol
- 5 minutes in 70% ethanol
- 5 minutes in 50% ethanol
- 5 minutes in distilled water
4. Block endogenous peroxidase by incubating section in 3% H2O2 in absolute methanol for 10 minutes at room temperature.
5. Rinse sections 3 x 5 minutes in PBS, pH 7.5.
6. For blocking, incubate sections in humidity chamber for 1 hour at room temperature in PBS containing 10% BSA.
7. Dilute primary antibody PAb to iNOS (Prod. No. ALX-210-504) 1:500 in PBS containing 10% BSA. Incubate sections with primary antibody overnight at 4°C in humidity chamber.
8. Rinse sections 3 x 5 minutes in PBS, pH 7.5.
9. Secondary antibody incubation: Zymed Histostain-SP Kit. Incubate sections in secondary antibody prepared according to manufacturer’s instructions for 30 minutes at RT in humidity chamber.
10. Rinse sections 3 x 5 minutes in PBS, pH 7.5.
11. Incubate sections in Streptavidin-peroxidase conjugate prepared according to manufacturer’s instructions for 10 minutes at RT in humidity chamber.
12. Rinse sections 3 x 5 minutes in PBS, pH 7.5.
13. To visualize, develop with AEC according to manufacturer’s instructions for 5-10 minutes.
14. Wash well with distilled water.
Note: The procedures listed above are intended only as a guide. Optimal conditions must be determined individually for each application. |
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Product Literature References
Alterations of nitric oxide synthase expression and activity during rat lung transplantation: M. Liu, et al.; Am. J. Physiol. Lung Cell. Mol. Physiol.
278, L1071 (2000),
Abstract;
Differential inducible nitric oxide synthase expression in systemic and pulmonary vessels after endotoxin: E.J. Pulido, et al; Am. J. Physiol. Regul. Integr. Comp. Physiol.
278, R1232 (2000),
Abstract;
Granulated metrial gland cells contain nitric oxide synthase during pregnancy in the rat: S.M. Sladek, et al.; Placenta
19, 55 (1998),
Abstract;
Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland: M. Onoda and H. Inano; J. Histochem. Cytochem.
46, 1269 (1998),
Abstract;
Specificity of antibodies to nitric oxide synthase isoforms in human, guinea pig, rat, and mouse tissues: W. Coers, et al.; J. Histochem. Cytochem.
46, 1385 (1998),
Abstract;
Experimental allergic encephalomyelitis in the rat is inhibited by aminoguanidine, an inhibitor of nitric oxide synthase: W. Zhao, et al.; J. Neuroimmunol.
64, 123 (1996),
Abstract;
Induction of nitric oxide synthase in the human cardiac allograft is associated with contractile dysfunction of the left ventricle: N.P. Lewis, et al.; Circulation
93, 720 (1996),
Abstract;
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