RTN4 Antibody

What is RTN4 and why are antibodies against it important for research?

RTN4 (Reticulon 4), also known as NOGO, is a membrane-bound protein that primarily resides in the endoplasmic reticulum (ER). It belongs to the reticulon family of proteins that share a common reticulon homology domain (RHD) . RTN4 is crucial in shaping tubular endoplasmic reticulum and has been extensively studied for its role as a neurite outgrowth inhibitor .

RTN4 antibodies are valuable research tools because:

• They enable detection of different RTN4 isoforms (NOGO-A, NOGO-B, NOGO-C) across various tissues

• They facilitate investigation of RTN4's involvement in neurodegeneration, cancer progression, and cellular morphology

• They allow visualization of RTN4's subcellular localization and interactions with other proteins

RTN4's diverse functions in neurobiology, cancer pathways, and cellular architecture make antibodies against it essential for multiple research disciplines.

What are the different isoforms of RTN4 and how can antibodies distinguish between them?

RTN4 exists in multiple isoforms generated through alternative splicing, with three major variants:

Distinguishing between these isoforms requires careful antibody selection:

• Isoform-specific antibodies: Some antibodies target unique N-terminal regions present only in specific isoforms (particularly NOGO-A)

• Pan-RTN4 antibodies: Antibodies targeting the shared RHD domain will detect all isoforms

• Validation techniques: Western blotting can confirm specificity by revealing bands at characteristic molecular weights (190-210 kDa for NOGO-A, 45-50 kDa for NOGO-B, and 22-25 kDa for NOGO-C)

When selecting an antibody, researchers should verify which epitope it targets and whether it recognizes all or specific isoforms based on the experimental question.

How do RTN4 antibodies differ in terms of host species, clonality, and applications?

RTN4 antibodies show considerable diversity in their properties, which affects their suitability for different experimental applications:

Dilution recommendations vary by application:

Each antibody should be titrated in the specific experimental system to obtain optimal results, as performance can be sample-dependent .

What are the optimal protocols for using RTN4 antibodies in Western blotting?

Western blotting with RTN4 antibodies requires careful attention to several parameters:

Sample preparation:

• Brain tissue is often used as a positive control for NOGO-A detection

• Cell lines like A375, HepG2, A549, and SH-SY5Y are validated for RTN4 detection

• Complete lysis buffers containing protease inhibitors are essential for full extraction

Technical considerations:

• Expected bands: Prepare to visualize bands at 190-210 kDa (NOGO-A), 45-50 kDa (NOGO-B), and/or 22-25 kDa (NOGO-C), depending on tissue/cell type

• Gel percentage: Use lower percentage gels (5-8%) for resolving high molecular weight NOGO-A

• Transfer conditions: Extended transfer times (>1 hour) may be necessary for complete transfer of high molecular weight isoforms

• Blocking: 5% non-fat milk in TBS is typically effective

• Antibody incubation: Most RTN4 antibodies perform optimally at 1:1000-1:8000 dilution in blocking buffer with overnight incubation at 4°C

• Detection: HRP-conjugated secondary antibodies with enhanced chemiluminescence typically provide sufficient sensitivity

Example protocol from validation studies:

• Load 50μg of protein per lane on 5-20% SDS-PAGE gel

• Run electrophoresis at 70V (stacking)/90V (resolving) for 2-3 hours

• Transfer to nitrocellulose membrane at 150mA for 50-90 minutes

• Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Incubate with rabbit anti-RTN4 antibody at 0.25-1μg/mL overnight at 4°C

• Wash with TBS-0.1% Tween 3 times (5 minutes each)

• Incubate with goat anti-rabbit IgG-HRP (1:10000) for 1.5 hours at room temperature

• Develop using an enhanced chemiluminescent detection kit

How should RTN4 antibodies be used for optimal immunohistochemistry and immunofluorescence results?

RTN4 detection in tissue sections and cultured cells requires specific methodological considerations:

Immunohistochemistry (IHC) protocol:

• Tissue preparation:

  • Fixed, paraffin-embedded sections work well for RTN4 detection

  • Positive controls include: brain tissue, testis, and prostate cancer tissue

• Antigen retrieval:

  • Heat-mediated antigen retrieval in TE buffer (pH 9.0) is recommended

  • Alternative: citrate buffer (pH 6.0) may be used

• Blocking and antibody application:

  • Block with 10% goat serum

  • Apply primary antibody at 1:50-1:500 dilution

  • Incubate overnight at 4°C

• Detection systems:

  • Biotinylated secondary antibody followed by Streptavidin-Biotin-Complex (SABC) with DAB as chromogen

  • Alternatively, polymer-based detection systems may be used

Immunofluorescence (IF/ICC) protocol:

• Cell/tissue preparation:

  • Validated cell lines include A549, SH-SY5Y, A375, and U20S cells

  • For cultured cells: fix with 4% paraformaldehyde and permeabilize with suitable buffer

• Blocking and antibody application:

  • Block with 10% goat serum

  • Apply primary antibody at 1:200-1:800 dilution

  • Incubate overnight at 4°C

• Detection and visualization:

  • Use fluorophore-conjugated secondary antibodies (e.g., DyLight®488 or DyLight®594)

  • Counterstain nuclei with DAPI

  • Visualize using appropriate filter sets on a fluorescence microscope

For both applications, include appropriate negative controls (primary antibody omission) and positive controls to validate specificity.

What approaches can be used to validate RTN4 antibody specificity?

Rigorous validation of RTN4 antibody specificity is crucial for reliable experimental outcomes. Multiple complementary approaches should be employed:

1. Molecular weight verification:

• Compare observed band sizes on Western blots to expected molecular weights

• RTN4 isoforms should appear at: 190-210 kDa (NOGO-A), 45-50 kDa (NOGO-B), 22-25 kDa (NOGO-C)

• Note that observed weights may differ from calculated weights (130 kDa)

2. Knockdown/knockout validation:

• Use RTN4 siRNA or shRNA to reduce expression in cell lines

• Compare antibody signal between control and knockdown samples

• Several publications have validated antibodies using this approach

3. Peptide competition assay:

• Pre-incubate antibody with immunizing peptide before application

• Signal should be significantly reduced or eliminated if antibody is specific

4. Multiple antibody concordance:

• Compare staining patterns using different antibodies targeting distinct RTN4 epitopes

• Consistent patterns across antibodies suggest specificity

5. Positive and negative control tissues/cells:

• Validate using tissues with known RTN4 expression profiles:

  • Brain tissue (high NOGO-A expression in oligodendrocytes)

  • SH-SY5Y, A549, A375, HepG2 cells (demonstrated RTN4 expression)

6. Cross-species reactivity:

• Test antibody on samples from multiple species (human, mouse, rat)

• Compare observed patterns with known species-specific expression profiles

7. Multiple detection methods:

• Verify consistent results across different techniques (WB, IHC, IF, etc.)

• Discrepancies between methods should be critically evaluated

Researchers should document all validation approaches in publications to strengthen the reliability of their findings.

Why might RTN4 antibodies show unexpected banding patterns or molecular weights in Western blots?

Unexpected banding patterns when using RTN4 antibodies are relatively common and may have several explanations:

Alternative splicing and isoforms:

• RTN4 exists in multiple isoforms (NOGO-A, -B, -C) with different molecular weights

• The canonical full-length protein has a calculated molecular weight of 130 kDa, but NOGO-A typically migrates at 190-210 kDa on SDS-PAGE

• Additional splice variants may contribute to unexpected bands

Post-translational modifications:

• Glycosylation can increase apparent molecular weight

• Phosphorylation, particularly through the AKT pathway, may alter migration

• Other modifications may occur and affect mobility

Proteolytic processing:

• RTN4 may undergo proteolytic cleavage in certain cell types or conditions

• Processing can generate fragments that are detected by antibodies targeting preserved epitopes

Technical factors:

• Sample preparation (heating conditions, lysis buffer composition)

• Gel percentage (inadequate separation of high molecular weight proteins)

• Transfer efficiency (incomplete transfer of large proteins)

Antibody cross-reactivity:

• Antibodies may cross-react with other reticulon family members (RTN1, RTN2, RTN3)

• Non-specific binding to unrelated proteins with similar epitopes

Troubleshooting approach:

• Verify antibody specificity using knockdown/knockout controls

• Optimize sample preparation (different lysis buffers, protease inhibitors)

• Adjust gel percentage and running/transfer conditions

• Test multiple antibodies targeting different RTN4 epitopes

• Consider tissue-specific expression patterns of different isoforms

How can non-specific background staining be minimized when using RTN4 antibodies in immunohistochemistry?

Non-specific background is a common challenge when using RTN4 antibodies in immunohistochemistry. Several strategies can help minimize this issue:

Optimization of fixation and antigen retrieval:

• Evaluate different fixatives (formalin, paraformaldehyde, alcohol-based)

• Test multiple antigen retrieval methods:

  • Heat-mediated retrieval in TE buffer (pH 9.0) is recommended for many RTN4 antibodies

  • Alternative: citrate buffer (pH 6.0)

• Optimize retrieval duration and temperature

Blocking improvements:

• Increase blocking time (1.5-2 hours)

• Test different blocking agents:

  • 10% normal serum from the same species as secondary antibody

  • 5% BSA

  • Commercial blocking reagents

• Add 0.1-0.3% Triton X-100 to blocking buffer for membrane permeabilization

Antibody optimization:

• Titrate primary antibody (1:50-1:500 for IHC)

• Use antibodies purified by antigen affinity chromatography

• Extend washing steps (3-5 times, 5-10 minutes each)

• Incubate at 4°C overnight rather than at room temperature

Detection system considerations:

• Evaluate different detection methods (polymer-based vs. avidin-biotin systems)

• Use secondary antibodies specifically validated for IHC

• Consider using amplification systems only when necessary

Sample-specific controls:

• Include no-primary-antibody controls

• Perform peptide competition assays to confirm specificity

• Use tissue known to be negative for RTN4 as control

• Include positive control tissues (brain, testis)

If background persists, consider using alternative RTN4 antibodies from different suppliers or those targeting different epitopes.

What considerations are important when detecting different RTN4 isoforms across diverse tissue types?

Detecting RTN4 isoforms across different tissues requires careful consideration of their distinct expression patterns and biochemical properties:

Isoform-specific expression patterns:

Tissue-specific optimization strategies:

• Brain tissue:

  • Rich in lipids: use specialized extraction buffers

  • Contains high NOGO-A: optimal for antibody validation

  • Consider region-specific expression differences

• Cancer tissues:

  • RTN4 expression correlates with survival in multiple cancer types

  • Expression levels vary significantly between tumor types

  • Consider using tumor and adjacent normal tissue comparisons

• Vascular tissues:

  • Primarily express NOGO-B

  • Important in vascular remodeling and endothelial cell migration

  • May require specific fixation protocols to preserve structure

• Cell lines for isoform detection:

  • A549, SH-SY5Y, A375, HepG2: validated for RTN4 detection

  • U-87MG: useful for NOGO-A detection

  • Select appropriate positive controls based on isoform of interest

Methodological adaptations:

• Western blotting: Consider gradient gels (5-20%) to resolve all isoforms simultaneously

• IHC/IF: Optimize antigen retrieval for each tissue type

• Sample preparation: Different extraction methods may be needed for membrane-associated vs. ER-localized fractions

• Antibody selection: Choose antibodies validated in specific tissues of interest

Understanding the tissue-specific expression profile of RTN4 isoforms is critical for experimental design and interpretation of results.

How can RTN4 antibodies be utilized in cancer research and what do expression patterns reveal about tumor biology?

RTN4 antibodies have emerged as valuable tools in cancer research, revealing important connections between RTN4 expression and cancer progression:

RTN4 expression and cancer prognosis:

Analysis of The Cancer Genome Atlas (TCGA) datasets has revealed that RTN4 expression inversely correlates with patient survival across multiple cancer types:

Groups with higher RTN4 expression show approximately 5 times higher risk compared to low-expression groups, suggesting RTN4 as a potential prognostic marker .

RTN4 knockdown effects on cancer phenotypes:

RTN4 antibodies have been essential in validating knockdown experiments that demonstrate:

• Reduced proliferation of cancer cells in vitro

• Smaller tumor xenografts in mice

• Altered lipid homeostasis

• Disrupted AKT signaling

• Changes in cytoskeletal stability

Methodological approaches:

• Tissue microarray analysis:

  • RTN4 antibodies can be used to screen large cohorts of patient samples

  • Correlate expression with clinical parameters and outcomes

• Mechanistic studies:

  • Combine RTN4 antibodies with other pathway markers (pAKT, cytoskeletal markers)

  • Use in co-immunoprecipitation to identify cancer-specific interaction partners

• Therapeutic implications:

  • RTN4 knockdown enhances paclitaxel cytotoxicity both in vitro and in vivo

  • Antibody detection can monitor RTN4 levels following experimental interventions

Technical considerations:

• Human prostate cancer tissue has been validated for RTN4 antibody staining

• Cancer cell lines (A549, A375, HepG2) are suitable models for studying RTN4 function

• Different isoforms may have distinct functions in cancer progression

RTN4 antibodies enable crucial investigations into cancer biology and potentially identify new therapeutic targets for intervention.

What insights have RTN4 antibodies provided into neurobiology and potential therapeutic applications?

RTN4 antibodies have been instrumental in elucidating the complex roles of RTN4/NOGO in neurobiology and exploring therapeutic applications for neurological conditions:

Neurobiological functions revealed through antibody-based studies:

• Axonal regeneration inhibition:

  • NOGO-A acts as a potent inhibitor of neurite growth and axonal regeneration

  • This inhibition occurs through interaction with NOGO receptors (RTN4Rs)

  • Antibody studies have visualized NOGO-A expression in oligodendrocytes of CNS white matter

• Synapse formation and neuronal morphology:

  • RTN4 receptors bind to BAI adhesion-GPCRs via thrombospondin type 1-repeat (TSR) domains

  • This interaction regulates:

    • Dendritic arborization

    • Axonal elongation

    • Synapse formation

    • Neural network activity

• Signaling mechanisms:

  • RTN4-BAI interaction involves unique glycoconjugates

  • C-mannosylation of tryptophan and O-fucosylation of threonine in BAI TSR-domains creates a high-affinity interface with RTN4 receptors

Therapeutic implications explored using antibodies:

Methodological applications:

• Visualization techniques:

  • Immunohistochemistry with RTN4 antibodies shows expression in oligodendrocytes

  • Immunofluorescence reveals subcellular localization in neurons and glia

• Functional studies:

  • Antibody blockade experiments to inhibit RTN4 function

  • Detection of changes in RTN4 expression during development and injury

• Therapeutic development:

  • Ozanezumab (anti-NOGO-A antibody) has been tested in clinical settings

  • Monitoring effects of interventions on RTN4 expression and signaling

RTN4 antibodies continue to be essential tools for understanding neurobiological mechanisms and developing potential therapies for conditions where axonal regeneration and neuroplasticity are impaired.

How can RTN4 antibodies be applied in studies of endoplasmic reticulum structure and function?

RTN4 plays crucial roles in shaping endoplasmic reticulum (ER) morphology, making RTN4 antibodies valuable tools for studying ER structure and function:

Fundamental ER functions of RTN4:

• Required for inducing formation and stabilization of ER tubules

• Regulates membrane morphogenesis in the ER by promoting tubular ER production

• Influences nuclear envelope expansion and nuclear pore complex formation

• Affects proper localization of inner nuclear membrane proteins

Advanced applications in ER biology research:

• ER tubule formation and maintenance:

  • Visualize RTN4 at regions of high membrane curvature

  • Track dynamics of tubule formation using live imaging with fluorescently-tagged antibodies

  • Co-localization studies with other ER-shaping proteins (REEP, atlastins, lunapark)

• ER-nuclear envelope interactions:

  • Investigate RTN4's role in nuclear pore complex formation

  • Study localization during cell division and nuclear envelope reassembly

  • Examine interactions with nuclear envelope proteins

• ER stress responses:

  • Monitor RTN4 distribution changes during ER stress

  • Correlate RTN4 levels with unfolded protein response markers

  • Investigate potential protective roles in ER homeostasis

Methodological approaches:

Technical optimization for ER studies:

• Sample preparation:

  • Gentle fixation to preserve ER structure (2-4% PFA)

  • Careful permeabilization (low detergent concentrations)

  • Consider subcellular fractionation to enrich ER membranes

• Antibody selection:

  • Choose antibodies targeting the reticulon homology domain (RHD) for ER studies

  • Validate ER localization with markers like calnexin or PDI

  • Confirm specificity in RTN4 knockdown cells

• Visualization strategies:

  • Combine with ER-specific dyes (ER-Tracker, BODIPY-FL thapsigargin)

  • Use multiple fluorescent channels to distinguish ER domains

  • 3D reconstruction to appreciate the complex ER network