NRCAM Antibody, HRP conjugated

What is the optimal application range for NRCAM antibody with HRP conjugation?

NRCAM antibody with HRP conjugation is optimized for Western Blotting (WB), Immunocytochemistry (ICC), and Immunofluorescence (IF) applications. The HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows in these applications. For Western blotting, this antibody typically detects a protein of approximately 160 kDa, corresponding to the full-length NRCAM protein . For immunofluorescence applications, using alternative conjugates such as FITC may be more appropriate depending on your detection system . When working with tissue sections, proper antigen retrieval techniques should be employed to ensure optimal binding specificity.

How should I validate the specificity of an anti-NRCAM antibody for my experimental system?

Validation of anti-NRCAM antibody specificity should follow a multi-step approach:

• Positive control tissues: Test the antibody on tissues known to express NRCAM, such as brain tissue from mouse, rat, or pig models .

• Western blot analysis: Confirm a single band at the expected molecular weight (~160 kDa) in positive control samples .

• Knockout/knockdown validation: Compare staining patterns between wild-type samples and those with reduced NRCAM expression through genetic manipulation.

• Cross-reactivity assessment: Evaluate the antibody's performance across species of interest. The extracellular domain-targeted antibody (AA 30-845) shows cross-reactivity with human, mouse, and rat samples due to high sequence homology (human: 91% identity with 753/822 amino acids identical to mouse) .

• Peptide competition: Perform blocking experiments with the immunizing peptide to confirm binding specificity.

What dilution ranges are recommended for NRCAM antibodies in different applications?

Optimal dilution ranges for NRCAM antibodies vary by application and specific antibody formulation:

Always perform a dilution series to determine the optimal concentration for your specific experimental conditions, as sample type and fixation methods can significantly impact antibody performance .

How can I differentiate between NRCAM splice variants in experimental systems?

Differentiating between NRCAM splice variants requires strategic experimental design:

• Targeted antibody selection: Use antibodies that recognize specific domains affected by alternative splicing. For instance, to detect the Δex5Δex19 NRCAM variant prevalent in pediatric high-grade gliomas (pHGGs), employ antibodies targeting the conformation created by the absence of these microexons .

• 3D structural considerations: The full-length NRCAM forms a "horseshoe" conformation with a proline-rich microexon 19-encoded amino acid sequence creating a sharp bend between Ig-like 6 domain and the first fibronectin type-III domain. In contrast, the Δex5Δex19 variant adopts a more open conformation, creating distinct epitopes for selective antibody targeting .

• RT-PCR analysis: Complement immunodetection with RNA-level analysis using primers flanking the alternatively spliced regions (microexons 5 and 19) to distinguish between variants.

• Selection of isoform-specific antibodies: For detection of the Δex5Δex19 NRCAM variant, consider using specialized monoclonal antibodies such as clone 3F8, which has demonstrated approximately 10-fold higher affinity for this variant compared to the full-length protein .

What are the considerations for using NRCAM antibodies in studies of nodes of Ranvier and demyelinating conditions?

When investigating nodes of Ranvier and demyelinating conditions with NRCAM antibodies, researchers should address these methodological considerations:

• Co-localization studies: NRCAM clusters at nodes of Ranvier alongside voltage-gated sodium channels. Design multiplex immunofluorescence assays that co-stain for NRCAM and sodium channels (e.g., Nav1.6) to confirm proper nodal localization .

• Developmental timing: NRCAM plays distinct roles during nodal development versus maintenance. During development, it's required for normal clustering of sodium channels at heminodes, though not essential for mature node formation . Timeline experiments may be necessary to distinguish these phases.

• Partner protein interactions: NRCAM functions with GLDN (gliomedin) to maintain NFASC (neurofascin) and sodium channel clusters at mature nodes . Design co-immunoprecipitation experiments to investigate these protein complexes using anti-NRCAM antibodies suitable for immunoprecipitation.

• Demyelination models: In demyelinating conditions, nodal architecture is disrupted. Compare NRCAM distribution in healthy versus demyelinated tissues to assess nodal integrity, using fixation conditions that preserve membrane protein localization.

• Schwann cell-axonal contact regions: NRCAM mediates cell-cell contacts between Schwann cells and axons . Use high-resolution microscopy techniques (STED, SIM) with NRCAM antibodies to visualize these interaction sites.

How should NRCAM antibodies be applied in cancer research, particularly for glioma studies?

When utilizing NRCAM antibodies in glioma research, consider these methodological approaches:

• Isoform discrimination: Recent research has identified the Δex5Δex19 NRCAM variant as prevalent in pediatric high-grade gliomas (pHGGs). Employ isoform-selective antibodies like the monoclonal 3F8 that specifically recognizes this variant with approximately 10-fold higher affinity than full-length NRCAM .

• Immunohistochemical profiling: Create tissue microarrays of glioma samples of varying grades and perform immunohistochemistry with anti-NRCAM antibodies to assess expression patterns. Compare staining intensity and localization with patient outcomes to identify potential prognostic correlations.

• Functional studies: For investigating the role of NRCAM in glioma cell migration and invasion, use neutralizing NRCAM antibodies in transwell migration assays or spheroid invasion models. The Δex5Δex19 NRCAM variant has been shown to be essential for pHGG cell migration, invasion, and tumor growth in vivo .

• Therapeutic targeting: For potential immunotherapeutic applications, antibodies like 3F8 can be used in conjunction with universal immune receptor (UIR) technologies to redirect T cell specificity toward cells expressing the Δex5Δex19 NRCAM variant. This approach has shown efficacy in killing pHGG cells expressing this variant while sparing cells with full-length NRCAM .

What are common causes of non-specific binding with NRCAM antibodies and how can they be mitigated?

Non-specific binding with NRCAM antibodies can compromise experimental results. Here are methodological solutions to common challenges:

• Background in neural tissues: Neural tissues often have high endogenous peroxidase activity that can cause background with HRP-conjugated antibodies. Implement a peroxidase quenching step (e.g., 3% hydrogen peroxide for 10 minutes) before antibody incubation.

• Cross-reactivity with related proteins: NRCAM shares approximately 50% identity with neurofascin . To minimize cross-reactivity:

  • Use antibodies specifically validated against multiple L1-IgCAM family members

  • Include appropriate negative controls (tissues/cells known to lack NRCAM but express related proteins)

  • Consider pre-adsorption with recombinant related proteins

• Binding to Fc receptors: Mouse monoclonal antibodies like the S364-51 clone may bind to Fc receptors on certain cell types. Include an Fc receptor blocking step using normal serum from the host species of your secondary antibody (or commercially available Fc block) before primary antibody incubation.

• Optimization of blocking conditions: For particularly challenging samples, test different blocking solutions (5% BSA, 5-10% normal serum, commercial blocking reagents) and extended blocking times (1-2 hours at room temperature).

How should I troubleshoot weak or absent signals when using HRP-conjugated NRCAM antibodies?

When facing weak or absent signals with HRP-conjugated NRCAM antibodies, implement this systematic troubleshooting approach:

• Antigen retrieval optimization:

  • For formalin-fixed paraffin-embedded tissues, compare heat-induced epitope retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Adjust retrieval time (10-30 minutes) and temperature

  • For membrane proteins like NRCAM, mild detergent treatment (0.1-0.3% Triton X-100) may improve accessibility

• Sample preparation considerations:

  • Ensure protein denaturation is complete for Western blotting

  • Fresh tissue preparation vs. frozen storage can affect epitope integrity

  • Fixation conditions greatly impact NRCAM detection; compare 4% PFA and acetone fixation

• Signal amplification strategies:

  • Implement tyramide signal amplification (TSA) for low-abundance detection

  • Increase antibody concentration incrementally

  • Extend primary antibody incubation time (overnight at 4°C)

  • Consider alternative detection substrates with higher sensitivity (e.g., SuperSignal West Femto for Western blotting)

• Verification of HRP activity:

  • HRP conjugates can lose activity over time or with improper storage

  • Test the HRP conjugate with a model substrate to confirm enzymatic activity

  • Consider switching to unconjugated primary antibody with separate HRP-conjugated secondary antibody if problems persist

How can NRCAM antibodies be utilized in studies investigating alternative splicing in neurological disorders?

NRCAM antibodies offer valuable tools for investigating alternative splicing in neurological disorders:

• Isoform-specific detection strategies: Use antibodies that recognize specific epitopes created by alternative splicing events. For example, antibodies targeting the conformational changes resulting from microexon 5 and 19 skipping can be used to profile NRCAM splicing patterns across different neurological conditions .

• Quantitative analysis workflow:

  • Perform Western blotting with antibodies recognizing common NRCAM domains

  • Analyze band patterns to identify potential isoforms

  • Follow up with isoform-specific antibodies to confirm variant identity

  • Integrate with RT-PCR data targeting alternative splicing junctions

• Spatial distribution mapping: Use immunohistochemistry or immunofluorescence with isoform-specific antibodies to map the spatial distribution of NRCAM variants in brain tissues from patients with neurological disorders compared to healthy controls.

• High-throughput screening applications: Develop ELISA-based systems using isoform-specific antibodies to screen patient samples for aberrant NRCAM splicing patterns that may serve as biomarkers for specific neurological conditions.

What considerations should be made when using NRCAM antibodies for developing targeted therapeutics in glioma?

For researchers using NRCAM antibodies in therapeutic development for glioma, these methodological principles should be followed:

• Selectivity verification: Rigorously validate antibody selectivity for tumor-specific NRCAM variants versus normal brain-expressed forms. The Δex5Δex19 NRCAM variant shows promise as a highly selective target for pHGG, with minimal expression in normal brain tissue .

• Cytotoxicity assessment protocol:

  • "Paint" target cells with the isoform-specific antibody (e.g., 3F8 for Δex5Δex19 NRCAM)

  • Co-culture with effector cells (e.g., T cells expressing CD64-based universal immune receptors)

  • Measure cell killing using flow cytometry with viability dyes or real-time cell analysis systems

  • Include appropriate controls: isotype control antibodies, untransduced effector cells, and target cells lacking the specific NRCAM variant

• Blood-brain barrier considerations: For in vivo applications, evaluate antibody size, charge, and lipophilicity to optimize blood-brain barrier penetration. Consider alternative delivery strategies such as convection-enhanced delivery or intranasal administration for improved target engagement.

• Off-target binding assessment: Comprehensive tissue cross-reactivity studies should be performed to identify potential off-tumor binding sites and predict adverse effects. This is particularly important for therapeutic applications targeting NRCAM variants.

What is the optimal protocol for generating and validating monoclonal antibodies against specific NRCAM isoforms?

Generating and validating monoclonal antibodies against specific NRCAM isoforms requires a systematic approach:

• Immunogen design and preparation:

  • Express the target NRCAM isoform in mammalian cells (e.g., NIH3T3 or CHO cells)

  • For cell-based immunization, ensure high surface expression through flow cytometry verification

  • For recombinant protein immunization, design constructs that preserve conformation-dependent epitopes

• Immunization and hybridoma generation:

  • Immunize syngeneic mice with cells expressing the target NRCAM isoform

  • Implement a 12+ week boosting protocol to maximize response

  • Isolate splenocytes from high-titer mice and electrofuse with appropriate myeloma cells (e.g., FOX-NY)

  • Culture and expand hybridomas in selective media

• Screening and selection workflow:

  • Primary screen: ELISA against recombinant target protein or flow cytometry with expressing cells

  • Secondary screen: Comparative binding analysis between target isoform and closely related variants

  • For NRCAM Δex5Δex19-selective antibodies, screen against cells expressing empty vector, full-length NRCAM, and Δex5Δex19 NRCAM

  • Select clones showing at least 10-fold binding preference for the target isoform

• Antibody purification and characterization:

  • Subclone positive hybridomas to ensure monoclonality

  • Determine antibody isotype and purify using appropriate affinity chromatography

  • Characterize by Western blot, flow cytometry, immunoprecipitation, and functional assays

  • Verify epitope specificity through peptide competition or epitope mapping

How should I optimize Western blotting protocols specifically for NRCAM detection?

Optimizing Western blotting for NRCAM detection requires attention to specific technical parameters:

• Sample preparation optimization:

  • For membrane proteins like NRCAM, use lysis buffers containing 1% NP-40 or Triton X-100

  • Include protease inhibitors to prevent degradation

  • Avoid excessive heating of samples (65°C for 5 minutes may be preferable to boiling)

  • Consider using gradient gels (4-12% or 4-15%) to resolve the full ~160 kDa protein

• Transfer conditions:

  • For large proteins like NRCAM (~160 kDa), use wet transfer methods

  • Extend transfer time (overnight at 30V/4°C) or use higher molecular weight transfer programs

  • Include SDS (0.1%) in transfer buffer to improve elution of large proteins from the gel

  • PVDF membranes typically perform better than nitrocellulose for large proteins

• Blocking and antibody incubation:

  • Test multiple blocking agents (5% non-fat milk vs. 5% BSA) as this can significantly impact background

  • For HRP-conjugated NRCAM antibodies, dilution ranges of 1:5,000-1:50,000 may be appropriate depending on expression levels

  • Extend primary antibody incubation time (overnight at 4°C) for optimal binding

  • Include 0.05-0.1% Tween-20 in wash buffers to reduce background

• Detection optimization:

  • For HRP-conjugated antibodies, select detection reagents appropriate for the expression level

  • Consider enhanced chemiluminescence substrates for improved sensitivity

  • Optimize exposure times to capture the signal within the linear range

How can NRCAM antibodies be incorporated into multiplex imaging platforms for neural circuit mapping?

Integrating NRCAM antibodies into multiplex imaging platforms requires strategic technical considerations:

• Antibody panel design for neural circuit mapping:

  • Combine NRCAM antibodies with markers for specific neuronal populations (NeuN, MAP2)

  • Include glial markers (GFAP, Iba1) to distinguish neuronal vs. glial components

  • Add markers for synaptic structures (Synaptophysin, PSD95) and myelination (MBP, MOG)

  • Select antibodies raised in different host species or use directly conjugated primary antibodies to avoid cross-reactivity

• Multiplexing methodologies:

  • Sequential immunostaining with stripping protocols (e.g., glycine-SDS buffer, pH 2.5)

  • Tyramide signal amplification (TSA) with heat-mediated antibody removal

  • Spectral unmixing of fluorophores with overlapping spectra

  • Mass cytometry (CyTOF) using metal-conjugated antibodies for highly multiplexed analysis

• Image acquisition and analysis workflow:

  • Apply deconvolution algorithms to improve spatial resolution

  • Implement automated image segmentation to identify specific cellular compartments

  • Use colocalization analysis to quantify spatial relationships between NRCAM and other markers

  • Consider machine learning approaches for pattern recognition in complex datasets

• Spatial context preservation:

  • For investigating nodes of Ranvier, use tissue preparation methods that preserve axonal architecture

  • Consider expansion microscopy techniques for improved spatial resolution of fine structures

  • Employ tissue clearing methods (CLARITY, iDISCO) for 3D visualization of NRCAM distribution in intact circuits

What are the best approaches for using NRCAM antibodies in single-cell proteomic analyses?

Single-cell proteomic analyses with NRCAM antibodies require specific methodological considerations:

• Sample preparation optimization:

  • For flow cytometry-based analyses, use gentle cell dissociation methods that preserve membrane proteins

  • Optimize fixation conditions (2-4% PFA, 10 minutes) to maintain epitope accessibility

  • Include viability dyes to exclude dead cells that may bind antibodies non-specifically

  • Consider cell surface protein isolation techniques for enrichment prior to analysis

• Single-cell mass cytometry (CyTOF) implementation:

  • Conjugate anti-NRCAM antibodies with rare earth metals

  • Include markers for cell identity, activation state, and functional parameters

  • Develop optimized staining protocols with shorter incubation times to minimize cell loss

  • Apply dimensionality reduction techniques (tSNE, UMAP) to visualize heterogeneity

• Advanced flow cytometry approaches:

  • Use fluorescence-activated cell sorting (FACS) with anti-NRCAM antibodies to isolate specific cell populations

  • Implement index sorting to link sorted cell phenotypes with downstream analyses

  • Apply spectral flow cytometry for increased parameter space with reduced compensation requirements

  • Consider imaging flow cytometry to combine phenotypic and morphological analyses

• Integration with spatial techniques:

  • Combine single-cell proteomics with spatial transcriptomics or in situ sequencing

  • Use DNA-barcoded antibodies for simultaneous protein and RNA detection

  • Apply computational approaches to integrate single-cell proteomic data with spatial information

  • Validate findings with traditional immunohistochemistry or immunofluorescence