NOMO1 Antibody

What is NOMO1 and what biological functions does it serve?

NOMO1 (Nodal Modulator 1) is a transmembrane protein that plays critical roles in developmental processes and stem cell maintenance. It functions as a modulator of signaling pathways involved in cell differentiation and tissue patterning, making it a key player in embryonic development and organogenesis. Research into NOMO1 has important implications for understanding developmental disorders, cancer progression, and regenerative medicine . Recent evidence suggests NOMO1 acts as a force-bearing transmembrane protein that diffuses slowly within the endoplasmic reticulum membrane, relying on specific immunoglobulin domain interfaces for proper function .

Which species do commercial NOMO1 antibodies typically react with?

Commercial NOMO1 antibodies demonstrate specific reactivity patterns across species. Most available antibodies show confirmed reactivity with human samples, while many also cross-react with mouse tissues. For example, Proteintech's NOMO1 antibody (17792-1-AP) has been specifically validated for both human and mouse samples across multiple applications . The Assay Genie CAB10510 NOMO1 Rabbit Polyclonal Antibody indicates broader reactivity, covering human, mouse, and rat samples . Researchers should carefully verify the species reactivity in product documentation for their specific experimental needs.

What is the expected molecular weight for NOMO1 in Western blot experiments?

NOMO1 is a relatively large protein with a calculated molecular weight of approximately 134 kDa based on its 1222 amino acid sequence. In experimental Western blot analyses, NOMO1 is typically observed at approximately 135 kDa . This slight difference between calculated and observed molecular weights may result from post-translational modifications affecting protein migration in SDS-PAGE gels. When interpreting Western blot results, researchers should expect to visualize NOMO1 as a distinct band at approximately 135 kDa.

What cell types and tissues are appropriate positive controls for NOMO1 experiments?

Based on validation data from multiple antibody sources, several cell types and tissues serve as reliable positive controls for NOMO1 detection:

Including these validated positive controls in experiments helps establish proper antibody function and provides reference expression levels for comparative analyses .

What are the optimal antibody dilutions for different NOMO1 detection techniques?

Optimal antibody dilutions vary by application and specific antibody product. The following table summarizes recommended dilutions for various applications with different NOMO1 antibodies:

These recommendations serve as starting points, and researchers should optimize dilutions for their specific experimental systems . Titrating antibodies across a range of concentrations is often necessary to determine the optimal signal-to-noise ratio for each application.

What are the recommended antigen retrieval methods for NOMO1 immunohistochemistry?

For optimal NOMO1 detection in paraffin-embedded tissues, specific antigen retrieval methods have been validated:

Primary recommendation:

• Use TE buffer (10 mM Tris, 1 mM EDTA) at pH 9.0 for heat-induced epitope retrieval

Alternative method:

• Citrate buffer at pH 6.0 can be used if the primary method does not yield satisfactory results

The retrieval process typically involves heating the slides in the chosen buffer using pressure cooking (121°C for 3-5 minutes), microwave treatment (maintaining sub-boiling temperature for 10-20 minutes after initial boil), or water bath immersion (95-99°C for 20-30 minutes) . The choice between these methods may depend on tissue type and fixation conditions, requiring optimization for specific experimental setups.

How should researchers troubleshoot non-specific bands in NOMO1 Western blots?

When troubleshooting non-specific bands in NOMO1 Western blots, researchers should implement a systematic approach:

• Optimize antibody dilution: Test a range of dilutions to find the optimal concentration that provides specific signal with minimal background. For example, with Proteintech's 17792-1-AP, try dilutions across the 1:500-1:2000 range .

• Improve blocking efficiency: Use 5% BSA or milk in TBS-T for blocking and antibody incubation to reduce non-specific binding.

• Enhance membrane washing: Increase the number and duration of washing steps with appropriate buffers (TBST/PBST) between antibody incubations.

• Modify transfer conditions: For this large protein (135 kDa), use wet transfer at lower voltage (30V) for an extended period (overnight at 4°C) with 10% methanol in transfer buffer.

• Include positive controls: Run lysates from validated positive controls (e.g., A431 cells, human brain tissue) alongside experimental samples for comparison .

• Validate with alternative antibodies: Cross-validate results using antibodies targeting different NOMO1 epitopes to confirm specific bands.

What protocols are recommended for successful NOMO1 immunoprecipitation?

For successful NOMO1 immunoprecipitation, the following protocol has been validated:

• Lysate preparation:

  • Harvest cells (A431 cells are validated for NOMO1 IP) and lyse in ice-cold lysis buffer containing mild detergents (e.g., 1% NP-40) and protease inhibitors

  • Clarify lysate by centrifugation at 14,000 × g for 10 minutes at 4°C

• Antibody binding:

  • Add NOMO1 antibody (0.5-4.0 μg depending on protein concentration) to 1.0-3.0 mg of cleared lysate

  • Incubate with gentle rotation overnight at 4°C

• Immunoprecipitation:

  • Add Protein A/G beads to the lysate-antibody mixture

  • Incubate with gentle rotation for 2-4 hours at 4°C

  • Collect beads by centrifugation and wash 3-5 times with lysis buffer

• Analysis:

  • Elute in Laemmli sample buffer, analyze by SDS-PAGE and Western blot

  • Include appropriate controls (IgG control, input lysate)

  • Probe with NOMO1 antibody at the recommended dilution (1:500-1:2000)

This protocol has been validated using A431 cells, which serve as a positive control for NOMO1 immunoprecipitation .

How does NOMO1 function in developmental signaling pathways?

NOMO1 plays multifaceted roles in developmental signaling pathways:

• Nodal pathway modulation: As its name suggests, NOMO1 modulates Nodal signaling, which is crucial for embryonic development, particularly in establishing left-right asymmetry, mesoderm and endoderm formation, and neural patterning .

• Structural functions: NOMO1 serves as a force-bearing transmembrane protein within the endoplasmic reticulum, with specific immunoglobulin (Ig) domains mediating important interactions. Research demonstrates that Ig 1-2 and Ig 10-11 domains can dimerize, with mutations in these regions preventing rescue of NOMO-depleted ER morphology .

• Stem cell regulation: NOMO1 participates in signaling networks that maintain stem cell populations, making it relevant for both developmental biology and regenerative medicine applications .

Understanding these developmental roles has significant implications for research into congenital disorders, tissue engineering, and cell-based therapies where developmental pathway manipulation is essential.

What evidence connects NOMO1 to cancer biology?

Several lines of evidence suggest potential connections between NOMO1 and cancer biology:

• Expression in cancer cells: NOMO1 has been detected in multiple cancer cell lines including:

  • A431 (human epidermoid carcinoma)

  • COLO 320 (human colorectal adenocarcinoma)

  • MCF-7 (human breast cancer cells)

  • NOMO-1 (acute myeloid leukemia cells)

• Association with leukemia: The NOMO-1 cell line is characterized as a CD74+ acute myeloid leukemia (AML) cell line. In studies with STRO-001 (an antibody-drug conjugate targeting CD74), these cells showed potent, target-dependent cytotoxicity with an IC-50 of 1.3 nm .

• Potential therapeutic implications: In xenograft models, STRO-001 therapy (3 weekly doses at 3 mg/kg) significantly inhibited leukemia growth in NSG mice transplanted with NOMO-1 cells, with 2 of 5 mice remaining disease-free until the study endpoint of 112 days .

• Expression in clinical samples: Immunohistochemistry has detected NOMO1 in human colon cancer tissue, suggesting potential relevance to colorectal cancer biology .

These findings suggest NOMO1 may have relevance to cancer biology, though more research is needed to establish causative relationships and potential therapeutic applications.

How can researchers study NOMO1 protein-protein interactions?

Several complementary techniques can effectively investigate NOMO1 protein-protein interactions:

• Co-immunoprecipitation (Co-IP): The 17792-1-AP NOMO1 antibody has been validated for immunoprecipitation in A431 cells, making it suitable for Co-IP experiments to identify NOMO1 interaction partners under native conditions .

• In vitro binding assays: Recombinant expression and purification of specific NOMO1 domains (such as Ig 1-2 and Ig 10-11) allows direct testing of domain interactions in controlled in vitro environments .

• Fluorescence Recovery After Photobleaching (FRAP): This technique has been successfully applied to study NOMO1 mobility within membranes, revealing how domain interactions (particularly the Ig 1/10/11 interface) influence protein diffusion properties .

• Structural modeling: Computational approaches like AlphaFold-Multimer have generated high-confidence models of NOMO1 domain interfaces, providing insights into potential interaction sites that can guide experimental design .

• Proximity labeling techniques: BioID or APEX2 fusions to NOMO1 could identify proximal proteins in the cellular environment, particularly valuable for membrane proteins like NOMO1.

For transmembrane proteins like NOMO1, approaches that preserve the native membrane environment are particularly informative for capturing physiologically relevant interactions.

What role does NOMO1 play as a force-bearing transmembrane protein?

Recent research has revealed NOMO1's function as a force-bearing transmembrane protein within the endoplasmic reticulum:

• Membrane diffusion characteristics: FRAP data demonstrates that NOMO1 diffuses slowly within the ER membrane, with this mobility dependent in part on the presence of specific immunoglobulin domain interfaces, particularly the Ig 1/10/11 interface .

• Domain interactions: Purified recombinant His6-Ig 1-2 and His6-Ig 10-11 domains demonstrate direct binding in vitro, with AlphaFold-Multimer generating high-confidence models of this interaction. Expression of these domains induces a dominant-negative void phenotype, suggesting their critical importance for NOMO1 function .

• ER morphology maintenance: NOMO1 contributes to maintaining endoplasmic reticulum morphology, as evidenced by the inability of NOMO4-Mut (a mutated form) to rescue NOMO-depleted ER morphology .

• Structural significance: The specific arrangement of NOMO1's immunoglobulin domains appears central to its force-bearing properties, with mutations disrupting these interactions affecting both protein mobility and cellular phenotypes .

These findings suggest NOMO1 plays important structural roles in the ER membrane, potentially by forming dimers or higher-order structures that contribute to organelle morphology and function.

How should researchers interpret variable NOMO1 expression across different tissues?

When interpreting variable NOMO1 expression across different tissues, researchers should consider multiple factors:

• Tissue-specific expression patterns: Based on validation data, NOMO1 shows differential expression across tissues, with detectable levels in human brain, colon, and pancreas tissues, as well as in specific cell lines . These variations likely reflect tissue-specific biological functions.

• Quantification approach: When comparing expression levels:

  • For Western blot: Normalize NOMO1 signal to appropriate loading controls

  • For IHC/IF: Consider both staining intensity and percentage of positive cells

  • For qPCR: Use validated reference genes for normalization

• Experimental variability: Perform multiple biological replicates to ensure reproducibility of observed expression differences.

• Developmental context: Given NOMO1's role in developmental processes, consider how expression patterns might correlate with tissue differentiation states or developmental stages .

• Disease relevance: Variations in expression between normal and pathological tissues (e.g., between normal colon and colon cancer) may have biological significance worth further investigation .

Systematic analysis across multiple tissue types using standardized methods can provide valuable insights into NOMO1's tissue-specific functions and potential disease associations.

What controls are essential for quantitative analysis of NOMO1 expression?

For rigorous quantitative analysis of NOMO1 expression, researchers should incorporate multiple controls:

• Positive controls:

  • For Western blot: Include A431 cells, COLO 320 cells, human brain tissue, or mouse colon tissue

  • For IHC: Include human colon tissue, human pancreas tissue

  • For IF: Include MCF-7 cells

• Negative controls:

  • K562 cells have been used as negative controls in some studies

  • Include secondary antibody-only controls to assess background

  • Use isotype controls to evaluate non-specific binding

• Loading/reference controls:

  • For Western blot: Include housekeeping proteins (β-actin, GAPDH) for normalization

  • For qPCR: Use validated reference genes appropriate for the tissue being studied

  • For IHC/IF: Include internal control tissues on the same slide

• Validation controls:

  • When possible, validate findings using multiple antibodies targeting different NOMO1 epitopes

  • Consider RNA interference approaches to confirm signal specificity

• Quantification standards:

  • Include a dilution series of positive control samples to ensure measurements fall within the linear range of detection

  • Use consistent image acquisition parameters across samples

Incorporating these controls enables reliable quantitative comparisons of NOMO1 expression across experimental conditions, tissues, or disease states.

How can researchers differentiate between NOMO1 isoforms or related family members?

Differentiating between NOMO1 isoforms or related family members requires careful experimental design:

• Antibody selection: Choose antibodies with well-defined epitopes. For example, the Novus Biologicals NBP2-46727 antibody was developed against a specific recombinant protein sequence (amino acids: GEKITVTPSSKELLFYPPSMEAVVSGESCPGKLIEIHGKAGLFLEGQIHPELEGVEIVISEKGASSPLITVFTDDKGAYSVGPLHSDLEYTVTS) , which may help differentiate between closely related proteins.

• Western blot resolution: Use gradient gels or lower percentage gels (6-8%) to achieve better separation of high molecular weight proteins, potentially resolving subtle size differences between isoforms.

• Multiple detection methods: Combine protein detection (Western blot) with mRNA analysis (RT-PCR, qPCR with isoform-specific primers) to distinguish between variants.

• Immunoprecipitation-mass spectrometry: This approach can definitively identify specific isoforms or family members based on peptide sequences unique to each variant.

• Reference databases: Compare experimental results with canonical sequences and known variants in protein databases.

• Recombinant standards: When available, include recombinant proteins representing specific isoforms as reference standards.

• Knockout/knockdown validation: Use genetic approaches (CRISPR, RNAi) targeting specific isoforms to confirm antibody specificity.

Understanding the specific NOMO1 isoform or family member present in experimental systems is critical for accurate interpretation of results and comparison across studies.

What factors might influence NOMO1 antibody sensitivity in different applications?

Multiple factors can influence NOMO1 antibody sensitivity across different applications:

• Epitope accessibility: As a transmembrane protein, NOMO1's epitopes may have variable accessibility depending on the application. The CAB10510 antibody targets amino acids 700-1000 of human NOMO1 , while the NBP2-46727 antibody targets a different sequence , potentially affecting accessibility in different experimental contexts.

• Sample preparation: For Western blot, complete protein denaturation is essential for exposing epitopes. For IHC, the specific antigen retrieval method significantly impacts detection - TE buffer at pH 9.0 is recommended as the primary method for NOMO1, with citrate buffer at pH 6.0 as an alternative .

• Fixation conditions: For immunostaining applications, overfixation can mask epitopes. Different fixatives (paraformaldehyde, methanol) may preserve different epitopes.

• Antibody format: The form of the antibody (unconjugated, HRP-conjugated, fluorophore-conjugated) can affect sensitivity and specificity.

• Detection systems: For IHC/IF, the sensitivity of various detection systems (direct fluorescence, amplified systems like tyramide signal amplification) can significantly impact results.

• Buffer composition: The presence of detergents, salts, and stabilizers in storage and incubation buffers can affect antibody binding efficiency.

• Target abundance: Applications involving rare proteins may require more sensitive detection methods or signal amplification.

Optimizing these factors for each specific application is essential for achieving reproducible and biologically meaningful results with NOMO1 antibodies.

How might NOMO1 function connect to muscular disorders?

Emerging research suggests potential connections between NOMO1 and muscular disorders:

• Expression in muscle tissue: There is evidence of NOMO1 expression in T. anterior muscle biopsies, with possible correlations between expression levels and dorsiflexion strength in control versus DM1 (presumably myotonic dystrophy type 1) patients .

• Force-bearing properties: NOMO1's characterization as a force-bearing transmembrane protein suggests potential mechanical functions that could be relevant to muscle physiology and pathology .

• Structural roles: NOMO1's role in maintaining ER morphology through specific domain interactions might have implications for muscle cells, where ER (sarcoplasmic reticulum) structure is critical for calcium handling and contractile function .

• Developmental signaling: As a modulator of developmental pathways, NOMO1 could influence muscle development, regeneration, or maintenance through effects on myogenic differentiation or satellite cell function .

These preliminary connections warrant further investigation to establish whether NOMO1 plays significant roles in normal muscle physiology or contributes to muscular disorders through mechanical, structural, or signaling mechanisms.

What therapeutic applications might target NOMO1 or use NOMO1 antibodies?

Several potential therapeutic applications involving NOMO1 or NOMO1 antibodies are suggested by current research:

• Cancer treatment: The NOMO-1 cell line (CD74+ AML) shows sensitivity to STRO-001, an antibody-drug conjugate. In xenograft models, STRO-001 therapy significantly inhibited leukemia growth, with some mice remaining disease-free . This suggests potential applications in targeting NOMO1-expressing cancer cells.

• Developmental disorders: Given NOMO1's role in developmental signaling pathways, therapeutic modulation might have applications in disorders resulting from developmental pathway dysregulation .

• Regenerative medicine: NOMO1's involvement in stem cell maintenance suggests potential applications in generating or maintaining stem cell populations for cell-based therapies .

• Diagnostic applications: NOMO1 antibodies might serve as diagnostic tools if expression patterns correlate with specific disease states, though this application requires further validation.

• Structural therapeutics: Understanding NOMO1's force-bearing properties and structural roles may lead to novel therapeutic approaches targeting mechanical aspects of cell function .

These potential applications remain largely theoretical and require substantial additional research to establish clinical relevance and therapeutic efficacy.

How does NOMO1 interact with the broader Nodal signaling pathway?

While the search results don't provide comprehensive details on NOMO1's interactions with the broader Nodal signaling pathway, several aspects can be inferred:

• Modulator function: As a Nodal Modulator, NOMO1 likely influences the Nodal signaling cascade, which typically involves:

  • Nodal ligand binding to type I and type II serine/threonine kinase receptors

  • Activation of Smad2/3 through phosphorylation

  • Complex formation with Smad4 and translocation to the nucleus

  • Regulation of target gene expression

• Developmental contexts: NOMO1's modulation of Nodal signaling is particularly relevant during embryonic development for processes including:

  • Left-right asymmetry determination

  • Mesoderm and endoderm formation

  • Neural patterning

• Potential mechanisms: NOMO1 might modulate Nodal signaling through:

  • Interaction with Nodal receptors or co-receptors

  • Influence on receptor trafficking or localization

  • Regulation of downstream signal transduction

  • Effects on signal termination or feedback mechanisms

Further research specifically investigating NOMO1's molecular interactions with Nodal pathway components would provide valuable insights into its precise modulatory mechanisms and potential therapeutic targeting.