NOS1 Antibody

What is NOS1 and what are its known alternative designations in scientific literature?

NOS1 (nitric oxide synthase 1) is a 161 kilodalton protein that may also be known by alternative nomenclature including bNOS, NC-NOS, nNOS, IHPS1, N-NOS, and nitric oxide synthase, brain. The protein plays critical roles in nitric oxide production across multiple tissue types, with significant functions in neuronal, muscular, and immune contexts . Understanding these designations is essential when searching literature and selecting appropriate antibodies for specific research applications.

What are the key considerations when selecting a NOS1 antibody for specific experimental applications?

When selecting a NOS1 antibody, researchers should consider:

• Target epitope specificity: Different antibodies target specific amino acid sequences (e.g., AA 9-136, AA 53-247) or post-translational modifications like phosphorylation sites (e.g., pSer852)

• Host species compatibility: Consider potential cross-reactivity issues if working with tissue from the same species as the antibody host

• Validated applications: Ensure the antibody has been validated for your specific application (e.g., WB, IHC, IP, ICC)

• Isoform specificity: Determine whether the antibody recognizes specific NOS1 isoforms or all variants

• Clonality: Monoclonal antibodies offer higher specificity while polyclonal antibodies may provide stronger signals through multiple epitope binding

How can researchers distinguish between different NOS1 isoforms using antibodies?

Distinguishing between NOS1 isoforms requires careful antibody selection and experimental design:

• Alpha (nNOS-α, 155 kDa) and mu (nNOS-μ, 160 kDa) isoforms can be distinguished by slight molecular weight differences in Western blotting

• Use isoform-specific antibodies targeting unique epitopes present in specific variants

• Brain tissue samples can serve as positive controls for alpha subtype, while cardiac tissue can be used for mu subtype validation

• Consider complementary approaches such as RT-PCR with isoform-specific primers to confirm antibody results

• When possible, use tissues from NOS1-knockout animals as negative controls to confirm specificity

What is the optimal protocol for detecting NOS1 via Western blotting?

For optimal NOS1 detection in Western blotting:

• Use cold cell lysis buffer with complete protease inhibitors

• Quantify total protein concentration with bicinchoninic acid assay

• Separate 30-50μg of total protein on 7-10% Bis-Tris SDS-polyacrylamide gels (critical for resolving the high molecular weight NOS1 protein)

• Transfer proteins onto PVDF membranes

• Block membranes with 5% nonfat dry milk for 1 hour

• Incubate with primary NOS1 antibody overnight at 4°C

• Incubate with HRP-conjugated secondary antibody for 2 hours

• Detect using enhanced chemiluminescent reagents

• Include appropriate positive controls (e.g., brain tissue homogenate)

What approaches are effective for studying protein-protein interactions involving NOS1?

Several approaches have proven effective for investigating NOS1 protein interactions:

• Co-immunoprecipitation (co-IP): Successfully used to demonstrate NOS1 interactions with partners like CAPON

  • Immunoprecipitate NOS1 using specific antibodies

  • Analyze precipitated complexes by immunoblotting with antibodies against suspected interaction partners

  • Include NOS1-deficient samples as negative controls

• Subcellular co-localization studies:

  • Dual immuno-gold electron microscopy has been used to demonstrate co-localization of NOS1 with proteins like CAPON in specific organelles (SR and mitochondria)

  • Sucrose gradient fractionation followed by Western blotting can identify compartment-specific interactions

How should researchers approach subcellular fractionation to study NOS1 localization?

Based on cardiac research, effective subcellular fractionation for NOS1 studies requires:

• Homogenization of tissue in appropriate buffer with protease inhibitors

• Sequential centrifugation to separate organelles

• Sucrose gradient ultracentrifugation for sarcoplasmic reticulum (SR) isolation (28%, 32%, 36%, and 40% gradients)

• Collection of purified SR fractions at specific gradient points (28% (#1) and 32% (#2))

• Verification of fraction purity using marker proteins (e.g., SERCA2 for SR)

• Protein concentration determination for each fraction prior to Western-blot experiments

What is the most effective approach for generating NOS1 knockout cell lines?

The CRISPR/Cas9 system has proven effective for generating NOS1-knockout cell lines:

• Design gRNAs targeting critical exons (e.g., exon six of NOS1 gene)

• Construct CRISPR/Cas9 plasmid with the designed gRNAs

• Transfect target cells and select with appropriate antibiotics (e.g., puromycin)

• Screen clones using PCR assay to identify potential knockout candidates

• Confirm knockout through Western blot to verify absence of protein expression

• Perform Sanger sequencing to characterize the specific mutations (e.g., deletions, insertions)

• Establish and maintain multiple confirmed knockout clones for experimental redundancy

How can researchers validate the functional consequences of NOS1 knockout in cancer models?

Based on melanoma research, comprehensive validation should include:

• In vitro assays:

  • Proliferation assessment (MTT assay, clonogenic assay, EdU assay)

  • Cell cycle analysis via flow cytometry (e.g., G0/G1 phase arrest in NOS1-knockout melanoma cells)

  • Gene expression profiling to identify differential expression genes (DEGs)

• In vivo models:

  • Tumor xenograft studies in appropriate mouse models (e.g., BALB/c-nu and C57BL/6)

  • Assessment of tumor growth and metastasis formation

  • Analysis of immune cell infiltration (e.g., CD3+ immune cells in tumors)

• Molecular pathway analysis:

  • Transcriptomics analysis with KEGG and CLUE GO pathway analysis

  • Validation of key regulated pathways (e.g., JAK-STAT and TOLL-LIKE pathways in melanoma)

What molecular pathways are affected by NOS1 inhibition or deletion in immune contexts?

NOS1 deletion or inhibition affects several key molecular pathways:

• Upregulation of interferon-stimulated genes (ISGs) in melanoma models

• Enhanced innate immune signaling, particularly JAK-STAT and TOLL-LIKE pathways

• Altered NF-κB transcriptional activity through mechanisms involving SOCS1

• Reduced inflammatory responses and tissue damage in experimental sepsis models

• Decreased tumor growth alongside increased infiltration of CD3+ immune cells in tumors

What are common challenges when detecting NOS1 via Western blotting and how can they be addressed?

Common challenges include:

• High molecular weight detection issues:

  • Use lower percentage gels (7-10%) to resolve the large 161 kDa protein

  • Optimize transfer conditions (time, buffer composition, voltage)

  • Consider wet transfer for more efficient transfer of large proteins

• Isoform differentiation:

  • Be aware that brain NOS1 (alpha subtype, 155 kDa) has slightly lower molecular weight than cardiac NOS1 (mu subtype, 160 kDa)

  • Use appropriate positive controls from relevant tissues

• Signal specificity concerns:

  • Include NOS1-knockout or knockdown samples as negative controls when possible

  • Validate antibody specificity using multiple antibodies targeting different epitopes

  • Consider immunoprecipitation followed by mass spectrometry for definitive identification

How can inconsistent NOS1 staining in immunohistochemistry be resolved?

Inconsistent IHC staining can be addressed through:

• Optimized fixation protocols:

  • Test different fixatives (paraformaldehyde, formalin) and fixation times

  • Evaluate various antigen retrieval methods (heat-induced vs. enzymatic)

  • Consider epitope accessibility issues in different tissues

• Antibody validation steps:

  • Test multiple antibodies against different epitopes

  • Include NOS1-knockout tissues as negative controls

  • Use tissues with known high NOS1 expression as positive controls

• Signal amplification strategies:

  • Explore polymer-based detection systems

  • Consider tyramide signal amplification for low-abundance targets

  • Optimize primary antibody concentration and incubation time/temperature

What approaches can help reconcile contradictory results in NOS1 functional studies?

When facing contradictory findings:

• Methodological standardization:

  • Directly compare antibodies, protocols, and experimental conditions

  • Standardize sample preparation and handling procedures

  • Use multiple detection methods to cross-validate findings

• Context consideration:

  • Evaluate cell/tissue-specific effects and microenvironmental influences

  • Assess potential compensatory mechanisms in knockout models

  • Consider temporal dynamics of NOS1 expression and activity

• Comprehensive controls:

  • Include both pharmacological inhibition (e.g., N-PLA) and genetic approaches

  • Perform rescue experiments with NOS1 re-expression

  • Use multiple model systems to establish consistency across experimental contexts

How does NOS1 interact with the NF-κB pathway in inflammatory responses?

Recent research has revealed sophisticated interactions between NOS1 and the NF-κB pathway:

• NOS1-derived nitric oxide promotes NF-κB transcriptional activity

• S-nitrosation of SOCS1 by NOS1-derived NO impairs its binding to p65 and targets SOCS1 for proteolysis

• NOS1−/− macrophages show increased SOCS1 protein and decreased levels of p65 protein compared with wild-type cells

• NOS1−/− mice demonstrate reduced cytokine production, lung injury, and mortality in sepsis models

• This mechanism appears to be independent of IκB degradation but instead affects maintenance of p65 protein levels

What is the role of NOS1 in cancer and how might it be therapeutically targeted?

NOS1's role in cancer, particularly melanoma, suggests potential therapeutic avenues:

How does the interaction between NOS1 and CAPON affect cardiac function?

The NOS1-CAPON interaction has important implications for cardiac function:

• Subcellular localization:

  • NOS1 and CAPON are co-enriched in cardiac sarcoplasmic reticulum (SR) fractions

  • They co-immunoprecipitate in whole heart lysates and co-localize in SR and mitochondria

• Response to cardiac injury:

  • Following myocardial infarction (MI), both NOS1 and CAPON redistribute to caveolae and co-localize with caveolin-3

  • This redistribution requires the formation of a complex with CAPON through PDZ-domain interactions

• Potential clinical significance:

  • Altered NOS1 localization in injured myocardium may represent an adaptive or maladaptive response

  • Understanding these mechanisms could identify new therapeutic targets for heart failure or post-MI remodeling

Data Table 1: NOS1 Antibody Applications and Characteristics