NMRAL1 Antibody

What is NMRAL1 and why is it significant in redox biology research?

NMRAL1 (NmrA-like family domain-containing protein 1, also known as HSCARG) functions as a redox sensor protein that undergoes structural reorganization and subcellular redistribution in response to fluctuating intracellular NADPH/NADP+ ratios. This dynamic behavior makes it a critical component in cellular redox homeostasis research. At low NADPH concentrations, NMRAL1 exists predominantly as a monomer that binds to argininosuccinate synthase (ASS1), an enzyme involved in nitric oxide synthesis. This binding inhibits ASS1 activity, reducing nitric oxide production and subsequently preventing apoptosis. Under normal NADPH conditions, NMRAL1 forms homodimers that each bind one NADPH molecule, which structurally masks the ASS1 binding site .

The protein demonstrates higher binding affinity for NADPH compared to NADP+, and this NADPH binding is essential for stable dimer formation. Researchers frequently investigate NMRAL1 due to its interactions with oxidized nicotinamide adenine dinucleotide (NAD+) levels, which affect various cellular responses . This redox-sensing capability positions NMRAL1 as a valuable target for studying cellular adaptation to metabolic and oxidative stress conditions.

What experimental techniques are compatible with commercially available NMRAL1 antibodies?

Commercial NMRAL1 antibodies have been validated for multiple experimental applications. The rabbit polyclonal antibody (such as ab272637) has demonstrated compatibility with several key techniques:

• Western blotting (WB): Effective at concentrations around 0.4 μg/mL for detecting NMRAL1 in human samples

• Immunohistochemistry on paraffin sections (IHC-P): Successfully used to visualize NMRAL1 in tissue sections

• Immunocytochemistry/Immunofluorescence (ICC/IF): Suitable for cellular localization studies

Most commercial antibodies are developed against human NMRAL1, specifically targeting recombinant fragments within the first 150 amino acids of the protein . When planning experiments, researchers should note that while many antibodies show high specificity for human samples, cross-reactivity with other species should be experimentally validated before proceeding with non-human models.

How should researchers optimize NMRAL1 detection in immunofluorescence studies?

For optimal NMRAL1 detection in immunofluorescence experiments, researchers should follow this methodological approach:

• Sample preparation: Fix cells or tissue sections with 4% paraformaldehyde, followed by permeabilization with 0.1% Triton X-100

• Blocking: Implement protein blocking using appropriate blocking buffer (typically 5-10% normal serum from the species of the secondary antibody)

• Antigen retrieval: For tissue sections, perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0), with microwave or pressure cooker heating

• Primary antibody incubation: Dilute anti-NMRAL1 antibody (typically 1:100-1:500, optimized for each antibody) and incubate overnight at 4°C

• Washing and secondary detection: Wash thoroughly with PBS-T, then incubate with fluorophore-conjugated secondary antibodies

• Counterstaining and mounting: Use DAPI for nuclear counterstaining and appropriate mounting medium to preserve fluorescence

When studying NMRAL1 translocation between cellular compartments, as often observed in response to redox changes, researchers should include appropriate subcellular markers (nuclear, cytoplasmic) to precisely track redistribution patterns. This approach has successfully demonstrated NMRAL1 translocation from cytoplasm to nucleus in diabetic cardiomyopathy models following STAMP2 overexpression .

What controls should be included when validating NMRAL1 antibody specificity?

Rigorous validation of NMRAL1 antibody specificity requires multiple control approaches:

• Positive controls: Include samples known to express NMRAL1 (human cell lines such as SH-SY5Y and HEK-293T have demonstrated reliable NMRAL1 expression)

• Negative controls:

  • Omit primary antibody incubation

  • Use isotype control antibodies

  • Test on samples where NMRAL1 has been knocked down via RNA interference

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide or recombinant NMRAL1 protein before application to samples

• Knockdown validation: Compare staining between wild-type samples and those with NMRAL1 knockdown (using shRNA or siRNA)

• Cross-validation: Compare results from multiple antibodies targeting different epitopes of NMRAL1

In Western blotting applications, researchers should verify that the detected band appears at the expected molecular weight (~33-35 kDa for NMRAL1) and include appropriate loading controls (β-actin is commonly used) .

How can researchers effectively study NMRAL1's role in neurodevelopment?

Investigating NMRAL1's neurodevelopmental functions requires specialized experimental approaches:

These approaches enable researchers to dissect NMRAL1's specific contributions to neural progenitor proliferation and differentiation decisions, critical processes implicated in neurodevelopmental disorders.

What methodologies can reveal NMRAL1's impact on dendritic spine morphology?

Studying NMRAL1's influence on dendritic spine morphology requires specialized neuronal imaging and analysis techniques:

• Primary neuronal culture preparation:

  • Harvest and culture primary neurons from embryonic rat or mouse brain

  • Transfect neurons with NMRAL1 knockdown constructs (shRNA) alongside fluorescent markers for visualization

• Spine imaging and analysis:

  • Fix neurons at appropriate developmental stage (DIV14-21 typically shows well-developed spines)

  • Perform immunostaining for dendritic markers and NMRAL1

  • Acquire high-resolution confocal z-stack images of dendritic segments

  • Analyze spine density (spines per unit dendritic length) and morphology (head width, neck length, stubby/thin/mushroom categorization)

• Data interpretation considerations:

  • Research has shown that Nmral1 knockdown results in significant decrease in dendritic spine density

  • This finding connects NMRAL1 dysfunction to synaptic abnormalities observed in schizophrenia

  • Researchers should categorize spine types and assess spine maturation when examining NMRAL1's role in synaptogenesis

When implementing these methods, researchers should maintain consistent image acquisition parameters and use automated analysis software with manual verification to ensure objective quantification of spine characteristics.

How can researchers investigate NMRAL1's role in transcriptional regulation?

To examine NMRAL1's influence on gene expression and transcriptional networks:

• Transcriptome analysis following NMRAL1 manipulation:

  • Perform RNA sequencing after NMRAL1 knockdown or overexpression

  • Identify differentially expressed genes (DEGs) using appropriate statistical thresholds (|fold change| > 1.5, p-adjusted < 0.01)

  • Validate key DEGs via qPCR

• Pathway and gene ontology enrichment analysis:

  • Apply GO (biological processes) analysis to DEGs

  • Focus on enriched categories relevant to research question (e.g., neurodevelopment processes)

  • Research has shown NMRAL1 knockdown affects genes involved in:

    • Gliogenesis

    • Head development

    • Nervous system development

    • Cell adhesion

    • Cell proliferation and migration

    • Synaptic transmission

• Integration with ChIP-sequencing:

  • Identify genomic regions directly bound by NMRAL1 or its interacting partners

  • Correlate binding events with transcriptional changes

  • Investigate co-occupancy with known transcription factors

This multi-layered approach allows researchers to distinguish between direct and indirect effects of NMRAL1 on gene expression, providing deeper insights into its regulatory mechanisms in different cellular contexts.

What techniques can distinguish between monomeric and dimeric NMRAL1 forms?

Distinguishing between NMRAL1's monomeric and dimeric forms is crucial for understanding its redox-sensing function. Researchers should employ these methodological approaches:

• Native PAGE and Western blotting:

  • Prepare samples without reducing agents or SDS

  • Run samples on native polyacrylamide gels alongside molecular weight markers

  • Transfer and probe with NMRAL1 antibodies

  • Monomeric NMRAL1 (~33-35 kDa) and dimeric NMRAL1 (~66-70 kDa) will migrate differently

• Crosslinking studies:

  • Treat cells or purified proteins with membrane-permeable crosslinkers (e.g., DSS, BS3)

  • Analyze by SDS-PAGE and Western blotting

  • Compare band patterns under different NADPH/NADP+ conditions

• Co-immunoprecipitation with ASS1:

  • NMRAL1 in its monomeric form binds ASS1

  • Immunoprecipitate ASS1 and probe for NMRAL1 co-precipitation

  • Higher co-precipitation indicates greater proportion of monomeric NMRAL1

• NADPH fluorescence monitoring:

  • Exploit NADPH's intrinsic fluorescence properties

  • Measure fluorescence changes upon binding to NMRAL1 under various conditions

  • Correlate with NMRAL1 dimerization state

These techniques can be combined to create a comprehensive profile of NMRAL1's oligomeric state under different cellular redox conditions, providing insight into its functional dynamics.

How can NMRAL1 antibodies be applied in schizophrenia research?

Investigating NMRAL1's role in schizophrenia pathogenesis requires several specialized approaches:

• Genetic variant analysis:

  • Study the functional variant rs2270363 in the NMRAL1 promoter region

  • Perform genotyping in case-control cohorts

  • The risk allele (A) of rs2270363 disrupts transcription factor binding (USF1, MAX, MXI1) to the E-box element of NMRAL1 promoter

• Expression studies in postmortem brain tissue:

  • Compare NMRAL1 protein and mRNA levels between schizophrenia patients and controls

  • Use immunohistochemistry and Western blotting with anti-NMRAL1 antibodies

  • Research has demonstrated significant downregulation of NMRAL1 in brains of schizophrenia patients compared to healthy controls

• Transcription factor binding analysis:

  • Perform electrophoretic mobility shift assay (EMSA) to assess how rs2270363 affects transcription factor binding

  • Conduct supershift assays using antibodies against specific transcription factors (USF1)

  • Research has shown the G allele has two binding bands while the A allele has one, with the G allele preferentially binding USF1

• Reporter gene assays:

  • Create constructs containing the NMRAL1 promoter with different rs2270363 alleles

  • Measure reporter activity in neuronal cell lines (SH-SY5Y, SK-N-SH)

  • Studies indicate the A (risk) allele confers significantly higher promoter activity compared to the G allele

These methodologies enable researchers to establish functional connections between NMRAL1 genetic variations, expression levels, and schizophrenia-related cellular phenotypes.

What approaches can reveal NMRAL1's functions in diabetic cardiomyopathy?

To investigate NMRAL1's role in diabetic cardiomyopathy, researchers should implement these methodological strategies:

• Diabetic cardiomyopathy model establishment:

  • Induce diabetes in rats via intraperitoneal STZ injection

  • Confirm diabetic status through glucose tolerance testing

  • Assess cardiac function via echocardiography and left ventricular catheterization

  • Evaluate cardiac interstitial fibrosis through histological staining

• NMRAL1 subcellular localization analysis:

  • Perform immunofluorescence staining with anti-NMRAL1 antibodies on cardiac tissue sections

  • Co-stain with nuclear markers to assess nuclear translocation

  • Research has demonstrated that STAMP2 overexpression promotes NMRAL1 translocation from cytoplasm to nucleus in diabetic hearts

• Signaling pathway investigation:

  • Analyze NF-κB pathway activation via Western blotting for phosphorylated p65

  • Assess insulin signaling components (IRS-1, Akt phosphorylation)

  • Studies show STAMP2 inhibits p65 phosphorylation through promoting NMRAL1 retranslocation

• Functional recovery assessment:

  • Measure improvements in glucose tolerance and insulin sensitivity

  • Evaluate alleviation of diastolic dysfunction and myocardial fibrosis

  • Correlate with NMRAL1 subcellular distribution changes

This integrated approach allows researchers to connect NMRAL1's subcellular dynamics with specific signaling pathways and functional outcomes in diabetic cardiomyopathy.

How can CRISPR-Cas9 technology enhance NMRAL1 functional studies?

CRISPR-Cas9 genome editing offers powerful approaches for investigating NMRAL1 function:

• NMRAL1 promoter editing:

  • Design sgRNAs targeting the NMRAL1 promoter region containing rs2270363

  • Create isogenic cell lines differing only in the rs2270363 allele

  • Compare NMRAL1 expression levels between edited lines

  • Research has confirmed that CRISPR-Cas9-mediated editing of the genomic region containing rs2270363 affects NMRAL1 expression

• NMRAL1 knockout generation:

  • Design sgRNAs targeting coding regions of NMRAL1

  • Generate complete NMRAL1 knockout cell lines or animal models

  • Analyze resulting phenotypes in terms of:

    • Cellular redox status

    • Neurodevelopmental processes

    • Response to oxidative stress

    • Transcriptomic changes

• Regulatory element mapping:

  • Implement CRISPRi (CRISPR interference) to systematically inhibit candidate regulatory regions

  • Identify critical cis-regulatory elements controlling NMRAL1 expression

  • Create functional maps of NMRAL1 regulation in different cell types

• Precise mutation introduction:

  • Engineer specific NMRAL1 mutations affecting protein function

  • Create models with altered NADPH binding capacity

  • Examine consequences for dimerization and ASS1 interaction

CRISPR-based approaches provide unprecedented precision in manipulating NMRAL1 and its regulatory elements, enabling researchers to establish direct causality between genetic variations, expression levels, and cellular phenotypes.

What techniques best characterize NMRAL1's protein-protein interactions?

Comprehensive analysis of NMRAL1's protein interaction network requires these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate NMRAL1 using validated antibodies

  • Identify co-precipitated proteins via Western blotting or mass spectrometry

  • Verify interactions through reciprocal Co-IP

  • Research has established ASS1 as a key NMRAL1 interaction partner when in monomeric form

• Proximity labeling techniques:

  • Express NMRAL1 fused to BioID2 or TurboID in relevant cell types

  • Activate proximity-dependent biotinylation

  • Purify biotinylated proteins and identify by mass spectrometry

  • Compare interaction profiles under different redox conditions

• Förster resonance energy transfer (FRET):

  • Create fluorescent protein fusions with NMRAL1 and candidate partners

  • Measure FRET efficiency in living cells

  • Monitor dynamic changes in interactions following redox perturbations

  • Quantify spatial distribution of interactions

• Yeast two-hybrid screening:

  • Use NMRAL1 as bait to screen for novel interaction partners

  • Validate hits in mammalian systems via Co-IP and functional assays

  • Identify interaction domains through deletion mapping

These complementary approaches enable researchers to build comprehensive interaction maps for NMRAL1, revealing how its interaction network changes in response to redox conditions and in disease states.