NOS3 Mouse mAb: Selection, Application & Protocol Guide

NOS3 Mouse mAb is a mouse monoclonal antibody targeting endothelial nitric oxide synthase (eNOS/NOS3), designed to deliver consistent and specific detection of NOS3 protein across a range of immunological applications in cardiovascular and vascular biology research.

Product SKU

Ab350580

Antibody Format

Mouse Monoclonal Antibody (mAb)

Formulation

PBS only (no carrier protein, no preservative)

Target Antigen

NOS3 (eNOS, Endothelial Nitric Oxide Synthase)

Host Species

Mouse

Reference / Source Code

absea#KC-3402

Overview of NOS3 and Its Biological Significance

NOS3, also known as endothelial nitric oxide synthase (eNOS), is a constitutively expressed enzyme primarily found in vascular endothelial cells. It catalyzes the conversion of L-arginine to L-citrulline, producing nitric oxide (NO), a critical signaling molecule involved in vasodilation, blood pressure regulation, angiogenesis, and inhibition of platelet aggregation.

Dysregulation of NOS3 activity is associated with cardiovascular diseases including hypertension, atherosclerosis, and endothelial dysfunction. As a result, NOS3 is a widely studied target in cardiovascular research, making reliable antibody tools essential for accurate experimental outcomes.

NOS3 Mouse mAb (Ab350580) provides researchers with a highly specific reagent for detecting and characterizing NOS3 protein expression, localization, and regulation in relevant experimental models.

Recommended Applications and Suggested Dilutions

NOS3 Mouse mAb is suitable for use in standard immunological detection techniques. The following applications are commonly associated with anti-NOS3 monoclonal antibodies of this class; researchers should validate conditions empirically for their specific experimental system:

• Western Blotting (WB): Recommended as a primary application for detecting NOS3 at its expected molecular weight of approximately 133–140 kDa. Optimize primary antibody dilution (typically 1:500–1:2000) according to sample type and detection system.
• Immunofluorescence (IF): Suitable for visualization of NOS3 localization in fixed cell or tissue sections. Titrate appropriately for signal-to-noise optimization.
• Immunohistochemistry (IHC): Can be applied to formalin-fixed paraffin-embedded (FFPE) or frozen tissue sections for spatial expression profiling.
• Immunoprecipitation (IP): May be used to pull down NOS3 protein complexes for downstream interaction studies.

Note: Specific validated dilutions and application data for Ab350580 should be confirmed with the product datasheet or by contacting Aladdin Scientific technical support.

Storage, Handling and Formulation Guidance

NOS3 Mouse mAb (Ab350580) is supplied in PBS only, without added carrier proteins (such as BSA) or preservatives (such as sodium azide). This formulation is particularly advantageous for applications where carrier proteins or azide may interfere, including direct conjugation, enzyme-linked assays, or cell-based functional studies.

Recommended storage conditions (general guidelines for PBS-formulated mAbs; confirm with official datasheet):

• Store at –20°C for long-term storage to maintain activity.
• Aliquot upon receipt to avoid repeated freeze-thaw cycles, which can reduce antibody performance.
• For short-term use (up to 1 month), storage at 4°C is acceptable.
• Avoid exposure to direct light, repeated freeze-thaw, and microbial contamination.

Handling Tips: Because the formulation contains no preservative, maintain strict aseptic technique when handling. Spin down briefly before opening to collect all liquid. Do not dilute working stocks until needed.

Troubleshooting Common Experimental Issues

When working with NOS3 Mouse mAb, researchers may encounter the following issues. The guidance below reflects best practices for monoclonal antibodies in a PBS-only formulation:

Issue	Possible Cause	Suggested Action
No or weak signal	Suboptimal antibody concentration; inadequate antigen retrieval (IHC)	Increase antibody concentration; optimize antigen retrieval method; verify positive control expression
High background	Over-incubation; insufficient blocking; secondary antibody cross-reactivity	Reduce primary antibody concentration or incubation time; increase blocking stringency; titrate secondary antibody
Non-specific bands (WB)	Sample overloading; non-specific binding	Reduce protein loading; increase wash stringency; confirm antibody species/isotype compatibility
Loss of signal after storage	Repeated freeze-thaw cycles; degradation without preservative	Aliquot and store at –20°C; avoid freeze-thaw; use within recommended timeframe

For application-specific optimization, contact Aladdin Scientific technical support with lot number and experimental details.

Experimental Protocol: Western Blotting with NOS3 Mouse mAb

The following is a general Western blotting protocol for use with NOS3 Mouse mAb (Ab350580). Optimize conditions empirically for your specific sample and system.

1. Sample Preparation: Lyse cells or homogenize tissue in RIPA buffer supplemented with protease inhibitors. Determine protein concentration (BCA or Bradford assay). Load 20–40 µg total protein per lane.
2. SDS-PAGE: Resolve samples on a 6–8% polyacrylamide gel to resolve NOS3 at ~133–140 kDa.
3. Transfer: Transfer to PVDF or nitrocellulose membrane using wet or semi-dry transfer at appropriate conditions.
4. Blocking: Block membrane with 5% non-fat dry milk or 5% BSA in TBST for 1 hour at room temperature. (Note: use BSA-based blocking if phospho-specific detection is required downstream.)
5. Primary Antibody Incubation: Dilute NOS3 Mouse mAb in blocking buffer (suggested starting dilution 1:1000; optimize as required). Incubate overnight at 4°C with gentle agitation.
6. Washing: Wash 3 × 10 minutes with TBST.
7. Secondary Antibody: Incubate with an appropriate HRP-conjugated anti-mouse IgG secondary antibody (1:5000–1:10000) for 1 hour at room temperature.
8. Washing: Wash 3 × 10 minutes with TBST.
9. Detection: Develop with ECL substrate and image using a chemiluminescence imaging system or X-ray film.
10. Expected Result: A band at approximately 133–140 kDa corresponding to NOS3.

Always include a positive control lysate (e.g., HUVEC or endothelial cell lysate) and a negative control (no primary antibody) to validate results.

Frequently asked questions

What species reactivity does NOS3 Mouse mAb (Ab350580) have?

The specific species reactivity of Ab350580 should be confirmed on the official product datasheet. Anti-NOS3 monoclonal antibodies of this class commonly show reactivity against human NOS3, with cross-reactivity to mouse and rat dependent on the epitope targeted. Please consult the Aladdin Scientific product page or technical support for confirmed reactivity data.

Why is the antibody supplied in PBS only, and does this affect how I should use it?

The PBS-only formulation means the antibody contains no carrier proteins (such as BSA) and no preservatives (such as sodium azide). This makes it ideal for applications sensitive to these additives, including direct fluorescent or enzymatic conjugation, functional cell-based assays, and some ELISA formats. However, because no preservative is present, strict aseptic handling and proper aliquoting are critical to prevent degradation.

What is the expected molecular weight of NOS3 detected by this antibody?

NOS3 (eNOS) is a well-characterized enzyme with a predicted molecular weight of approximately 133 kDa; it may appear as a band between 130–140 kDa on Western blots due to post-translational modifications. Always confirm with a validated positive control such as HUVEC cell lysate.

How should I aliquot and store NOS3 Mouse mAb to maintain performance?

Upon receipt, centrifuge the vial briefly, then aliquot the antibody into single-use volumes to avoid repeated freeze-thaw cycles. Store aliquots at –20°C for long-term use. For frequent use within a month, a working aliquot may be kept at 4°C. Avoid exposure to light and do not add azide to the PBS-only formulation unless your application permits.

Can NOS3 Mouse mAb be used for immunoprecipitation or flow cytometry?

NOS3 Mouse mAb may be applicable to immunoprecipitation for pulling down NOS3 protein complexes. Suitability for flow cytometry depends on whether the target epitope is accessible under the chosen fixation and permeabilization conditions used. Researchers should validate these applications empirically and refer to the product datasheet or contact Aladdin Scientific technical support for confirmed application data for Ab350580.

References

1. Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012;33(7):829-837. doi:10.1093/eurheartj/ehr304
2. Dudzinski DM, Michel T. Life history of eNOS: partners and pathways. Cardiovasc Res. 2007;75(2):247-260. doi:10.1016/j.cardiores.2007.03.023
3. Aladdin Scientific. NOS3 Mouse mAb (Ab350580) Product Information. absea#KC-3402. Aladdin Scientific.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.