NRCAM Antibody, FITC conjugated

What is NrCAM and what cellular functions does it mediate?

NrCAM is a transmembrane cell adhesion molecule belonging to the L1 family of immunoglobulin superfamily (IgCAMs). It contains both immunoglobulin (Ig) and fibronectin type III (FNIII) domains in its extracellular region. NrCAM plays crucial roles in promoting axon growth and repulsion through trans-homophilic binding of its extracellular domains and intracellular coupling to the actin cytoskeleton through adaptors like ankyrin B and ezrin-radixin-moesin proteins . Additionally, NrCAM controls dendritic spine densities, axonal guidance and targeting, and neurite outgrowth by functioning as a co-receptor molecule at the neuronal cell surface . The protein is highly expressed in the cortex and contains a PDZ binding motif that potentially binds to PSD scaffold proteins like PSD-95 and SAP102 .

What epitope is recognized by Anti-NrCAM (extracellular)-FITC Antibody?

The Anti-NrCAM (extracellular)-FITC Antibody (product #ANR-045-F) is generated against a specific peptide sequence (C)KDNGELPNDERFSVD, corresponding to amino acid residues 583-597 of rat NrCAM (Accession P97686) . This epitope is located in the extracellular N-terminus of the protein, making this antibody particularly useful for detecting NrCAM on the surface of intact cells without permeabilization .

What expression patterns of NrCAM can be detected in neural tissues?

NrCAM displays distinct expression patterns across neural tissues. Immunohistochemical studies show that NrCAM is prominently expressed in the rat hippocampal dentate gyrus region, with immunoreactivity detected in neurons of the hilus . In lens tissue, NrCAM antibodies stain the cell surface of secondary fiber cells in both mouse and chick samples, showing regular staining patterns dependent on the direction and position of the section . Cross-sections reveal hexagonal fiber cells with differential staining intensity between radial and tangential cell surfaces .

How can NrCAM-FITC antibodies be applied in flow cytometry?

For direct flow cytometry applications with FITC-conjugated NrCAM antibodies:

• Prepare single-cell suspensions from your tissue of interest or use established cell lines

• Wash cells and adjust to 1×10⁶ cells per sample in flow cytometry buffer

• Add Anti-NrCAM (extracellular)-FITC Antibody at the appropriate concentration (2.5-5 μg per sample, depending on cell type)

• Include appropriate controls:

  • Unstained cells (autofluorescence control)

  • Isotype control-FITC (e.g., Rabbit IgG Isotype Control-FITC)

• Incubate for 30-45 minutes at 4°C in the dark

• Wash cells twice with flow buffer

• Analyze by flow cytometry in the FITC channel

This methodology has been validated for detecting NrCAM on intact mouse P815 mastocytoma cells (using 2.5 μg antibody) and human MEG-01 megakaryoblastic leukemia cells (using 5 μg antibody) .

What controls should be included for reliable flow cytometry analysis?

For rigorous flow cytometry analysis using FITC-conjugated NrCAM antibodies, include the following controls:

These controls help distinguish specific NrCAM binding from background signals and non-specific interactions, ensuring reliable interpretation of flow cytometry data .

How should samples be prepared for optimal NrCAM detection in tissue sections?

For effective immunohistochemical staining of NrCAM in tissue sections:

• Prepare perfusion-fixed frozen tissue sections or appropriately fixed cell cultures

• For tissue retrieval, use TE buffer (pH 9.0) or alternatively citrate buffer (pH 6.0)

• Block with appropriate serum (typically 5-10% normal goat serum)

• Apply Anti-NrCAM (extracellular) Antibody at 1:200 dilution

• Incubate overnight at 4°C

• Wash thoroughly with PBS

• Apply fluorophore-conjugated secondary antibody (e.g., goat anti-rabbit-AlexaFluor-488)

• Counter-stain nuclei with DAPI if desired

• Mount with anti-fade mounting medium

This protocol has been validated for detecting NrCAM in rat hippocampal dentate gyrus regions, where it shows clear neuronal immunoreactivity .

What are the recommended dilutions for different applications?

These dilutions should be optimized for your specific experimental conditions and sample types .

How does NrCAM couple to the cytoskeleton during axonal growth?

NrCAM coupling to the cytoskeleton is a complex process involving multiple protein domains and interactions:

• The cytoplasmic tail of NrCAM contains binding sites for cytoskeletal adaptor proteins, including ankyrin B

• NrCAM's FNIII domains facilitate cis-interactions with other membrane proteins

• Lipid raft partitioning plays a critical role in the actin-dependent retrograde movement of NrCAM

Experimental evidence using optical tweezers and single particle tracking of beads coated with NrCAM ligands (e.g., TAG-1) demonstrated that deletion of the cytoplasmic tail alone does not prevent coupling to the actin flow. Additional deletion of the FNIII domains is needed to abolish rearward movement, suggesting cooperative mechanisms for cytoskeletal coupling .

The actin-dependent retrograde movement of NrCAM also requires partitioning into lipid rafts, as demonstrated by cholesterol depletion experiments using methyl-β-cyclodextrin (MBCD). Adhesive contacts promote the recruitment of raft components like caveolin-1 to NrCAM clusters, suggesting that ligand binding induces the coalescence of lipid rafts, which contributes to cytoskeletal anchoring .

What experimental approaches can be used to study NrCAM-cytoskeleton interactions?

Researchers investigating NrCAM-cytoskeleton interactions can employ several sophisticated approaches:

• Optical tweezers with ligand-coated beads: This technique involves coating microbeads with NrCAM ligands like TAG-1 and using optical tweezers to manipulate these beads on the cell surface. By tracking bead movement, researchers can analyze the coupling between NrCAM and the actin cytoskeleton under various conditions .

• Single particle tracking: This approach enables visualization of individual NrCAM molecules or clusters and their dynamics on the cell surface. It can be combined with pharmacological treatments (e.g., cytoskeletal disruptors) to assess cytoskeletal dependencies .

• Fluorescence recovery after photobleaching (FRAP): FRAP experiments can measure the lateral mobility of NrCAM in different membrane domains, providing insights into its interactions with the cytoskeleton and other membrane components .

• Domain deletion mutants: Creating NrCAM constructs with specific domain deletions (e.g., cytoplasmic tail deletion, FNIII domain deletions) helps identify the domains crucial for cytoskeletal interactions. These constructs can be expressed in cell lines for functional studies .

• Lipid raft disruption: Treatments with agents like methyl-β-cyclodextrin (MBCD) that deplete cholesterol can assess the role of lipid rafts in NrCAM-cytoskeleton coupling .

Why might I experience weak or no signal when using NrCAM-FITC antibodies?

Several factors could contribute to weak or absent signals when using NrCAM-FITC antibodies:

• Low target expression: NrCAM expression varies across tissues and cell types. While it is highly expressed in the cortex, expression may be lower in other regions or cell populations .

• Improper fixation: Overfixation can mask epitopes, while underfixation may result in poor tissue preservation. For the extracellular epitope recognized by ANR-045-F, light fixation (e.g., 4% paraformaldehyde for 10-15 minutes) is often optimal .

• Insufficient antigen retrieval: For fixed tissues, appropriate antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0) may be necessary to expose the epitope .

• Antibody concentration: Using too low a concentration of antibody may result in weak signals. For flow cytometry, 2.5-5 μg per 10⁶ cells is recommended, depending on the cell type .

• Photobleaching: FITC is susceptible to photobleaching. Minimize exposure to light during preparation, and use anti-fade mounting media for microscopy applications.

• Sample degradation: Protein degradation during sample preparation can reduce antigen availability. Use fresh samples when possible and include protease inhibitors in lysis buffers.

How can I validate the specificity of NrCAM antibody staining?

To ensure the specificity of NrCAM antibody staining, implement these validation approaches:

• Blocking peptide control: Pre-incubate the antibody with NrCAM extracellular blocking peptide before application to samples. This should abolish specific staining, as demonstrated in rat hippocampus sections .

• Knockout/knockdown controls: When available, tissues or cells with genetic ablation of NrCAM provide excellent negative controls. A complete absence of signal in these samples confirms antibody specificity .

• Western blot correlation: Perform Western blots alongside immunostaining to confirm that the detected protein is of the expected molecular weight for NrCAM .

• Multiple antibody approach: Use antibodies recognizing different epitopes of NrCAM and compare staining patterns.

• Expression pattern consistency: Compare your staining pattern with published literature on NrCAM distribution. For example, specific staining in the rat hippocampal dentate gyrus region should show immunoreactivity in neurons of the hilus .

How can NrCAM antibodies contribute to autism spectrum disorder research?

NrCAM has been genetically linked to autism spectrum disorders (ASD), making it a valuable target for ASD research. Genetic polymorphisms in the NrCAM gene are associated with ASD, intellectual disability, and addictive behavior . NrCAM knockout mice demonstrate behaviors that align with ASD phenotypes, including:

• Impaired sociability

• Cognitive inflexibility

• Hypersensitivity to sensory stimuli

• Altered visual cortical responses

Researchers can use FITC-conjugated NrCAM antibodies to:

• Compare expression patterns: Investigate differences in NrCAM expression and localization between neurotypical subjects and those with ASD using immunohistochemistry or flow cytometry.

• Examine structural abnormalities: Study potential alterations in neuronal connectivity and synaptic organization that may be associated with NrCAM dysfunction.

• Evaluate developmental trajectories: Track NrCAM expression throughout neurodevelopment to identify critical periods where abnormalities might emerge.

• Test therapeutic interventions: Assess whether therapeutic approaches affect NrCAM expression or localization in animal models of ASD.

By combining NrCAM antibody applications with genetic analyses and behavioral assessments, researchers can gain deeper insights into the role of NrCAM in ASD pathophysiology and potentially identify new therapeutic targets.

What role does NrCAM play in axonal guidance, and how can this be studied?

NrCAM plays critical roles in axonal guidance and targeting through both homophilic and heterophilic interactions. Its Ig domains mediate binding to multiple ligands, enabling diverse signaling outcomes that guide growing axons .

Methodological approaches to study NrCAM in axonal guidance include:

• Growth cone dynamics: Track NrCAM-mediated retrograde movement on growth cones using ligand-coated microspheres, as demonstrated with F3/contactin-coated beads on cerebellar granule cells .

• Domain-specific functions: Use transfection of NrCAM deletion mutants (e.g., NrCAMΔfnΔCter) to determine which domains are essential for proper axonal guidance .

• Cytoskeletal interactions: Investigate how NrCAM couples to the actin cytoskeleton through ankyrin B and ezrin-radixin-moesin adaptors, and how this coupling affects growth cone motility .

• Lipid raft involvement: Examine the role of lipid rafts in NrCAM function by using cholesterol depletion (e.g., with MBCD) and assessing effects on NrCAM mobility and signaling .

• Knockout/knockdown studies: Compare axonal pathfinding in normal versus NrCAM-deficient models to identify guidance defects, as seen in studies of lens fiber organization in NrCAM-deficient mice .

By combining these approaches, researchers can elucidate the molecular mechanisms through which NrCAM contributes to precise axonal pathfinding during neurodevelopment.