NCAM2 Antibody, FITC conjugated

What is NCAM2 and why is it important in neuroscience research?

NCAM2 is a member of the neural cell adhesion molecule family that plays important roles in selective fasciculation and zone-to-zone projection of the primary olfactory axons . It contains five immunoglobulin-like domains, two Fibronectin type III domains, a transmembrane domain, and a cytoplasmic domain . NCAM2 is expressed predominantly in adult and fetal brain tissue, making it significant for studies of neural development, axon guidance, and synapse formation. The protein's involvement in cell-to-cell interactions during growth suggests its crucial role in embryogenesis and development .

What are the key structural characteristics of NCAM2?

NCAM2 has an ectodomain consisting of 5 Ig domains followed by 2 membrane-proximal FnIII domains . The FnIII domains form a rigid structure with very low flexibility as demonstrated by small angle X-ray scattering (SAXS) data . Unlike NCAM1, the NCAM2 FnIII2 domain contains a Walker A motif that does not bind ATP, as shown through NMR spectroscopy and titration with ATP analogs . The protein has a predicted molecular weight of approximately 93 kDa .

How do I select the appropriate NCAM2 antibody for my research?

When selecting an NCAM2 antibody, consider the following factors:

• Target species: Verify the antibody's reactivity with your species of interest. For example, the FITC-conjugated NCAM2 polyclonal antibody (bs-11094R-FITC) reacts with human samples and is predicted to react with mouse, rat, and rabbit samples .

• Application compatibility: Ensure the antibody is validated for your intended application. NCAM2 antibodies are available for various applications including Western Blot (WB), Immunohistochemistry (IHC), Flow Cytometry (FCM), Immunofluorescence (IF), and ELISA .

• Epitope location: Consider which region of NCAM2 you need to target. For instance, bs-11094R-FITC targets an epitope within amino acids 51-150 of the 837 amino acid human NCAM2 protein .

• Conjugation: Select antibodies with appropriate conjugates for your detection method. FITC-conjugated antibodies are particularly useful for flow cytometry and fluorescence microscopy .

What is the recommended protocol for using NCAM2 antibody in flow cytometry?

For flow cytometry applications using FITC-conjugated NCAM2 antibody:

• Sample preparation:

  • Harvest cells (1-5 × 10^6) and wash twice with PBS

  • Fix cells in 4% paraformaldehyde for 10 minutes at room temperature if intracellular staining is required

  • Permeabilize with 0.1% Triton X-100 if detecting intracellular antigens

• Staining procedure:

  • Block non-specific binding with 5% normal serum for 30 minutes

  • Dilute NCAM2 antibody (bs-11094R-FITC) at 1:20-1:100 in blocking buffer

  • Incubate cells with diluted antibody for 30-60 minutes at room temperature in the dark

  • Wash cells 3 times with PBS

  • Analyze by flow cytometry with appropriate filters for FITC detection (excitation ~495 nm, emission ~520 nm)

• Controls:

  • Include an isotype control (FITC-conjugated rabbit IgG)

  • Include unstained cells for autofluorescence baseline

How does the binding mechanism of NCAM2 to FGFR differ from that of NCAM1?

The NCAM2 FnIII domains form a rigid structure that binds and activates FGFR in a manner related to, but distinct from NCAM1 . While both NCAM1 and NCAM2 FnIII2 domains contain Walker A motifs, NMR spectroscopy and titration experiments revealed that unlike NCAM1, the NCAM2 Walker A motif does not bind ATP . This fundamental difference affects how each molecule interacts with FGFR.

In NCAM1, ATP binding to the Walker A motif interferes with FGFR binding. Since NCAM2 does not bind ATP at this site, its interaction with FGFR is not modulated by ATP levels . Additionally, SAXS data has shown that the NCAM2 FnIII1-2 double domain exhibits remarkably low flexibility, creating a rigid binding interface for FGFR interaction . This structural rigidity may contribute to the specificity and strength of NCAM2-FGFR binding.

What are the methodological considerations for optimizing NCAM2 immunohistochemistry with FITC-conjugated antibodies?

Optimizing NCAM2 immunohistochemistry with FITC-conjugated antibodies requires addressing several technical considerations:

• Tissue preparation:

  • For paraffin-embedded tissues: Complete deparaffinization and rehydration, followed by heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • For frozen sections: Fix in cold acetone or 4% paraformaldehyde before staining

• Signal optimization:

  • Titrate antibody concentration (recommended starting dilution for bs-11094R-FITC: 1:20-1:100)

  • Include antigen retrieval optimization experiments using different buffers and pH conditions

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

  • Use Sudan Black B (0.1% in 70% ethanol) to reduce autofluorescence in neural tissues

• Multiplex considerations:

  • When performing multiplex staining, select additional fluorophores with minimal spectral overlap with FITC

  • Perform sequential staining for multiple rabbit-derived antibodies to avoid cross-reactivity

  • Include appropriate controls for spectral unmixing

• Validation controls:

  • Positive control: Human brain tissue (high NCAM2 expression)

  • Negative control: Tissue known to have minimal NCAM2 expression

  • Isotype control: FITC-conjugated rabbit IgG at the same concentration

A case study from paraffin-embedded human kidney tissue demonstrated successful NCAM2 staining using a concentration of 20 μg/ml, which may serve as a reference point for optimization .

How can I troubleshoot weak or non-specific signals in Western blots using NCAM2 antibodies?

Troubleshooting weak or non-specific signals in Western blots requires systematic evaluation of multiple parameters:

When blotting for NCAM2, Western blot data indicates that a concentration of 1 μg/mL of antibody is effective for detecting the predicted 93 kDa band in both pig and rat brain lysates, as well as in recombinant human NCAM2 protein samples . Guinea pig anti-rabbit HRP-linked secondary antibody at 1:2000 dilution has been successfully used for detection .

What is the role of Stat5 in regulating NCAM2 expression and how can this be studied experimentally?

Stat5 has been identified as a regulator of NCAM2 expression through its binding to the NCAM2 intron in the NKL natural killer cell line . This binding is specifically induced by cytokines that activate Stat5. Interestingly, neither Stat1 nor Stat3 bind to this region despite sharing a consensus binding sequence with Stat5 .

To experimentally investigate this regulatory mechanism:

• Chromatin Immunoprecipitation (ChIP):

  • Cross-link protein-DNA complexes in cytokine-stimulated cells

  • Immunoprecipitate with anti-Stat5 antibodies

  • Analyze NCAM2 intronic regions by qPCR or sequencing

  • Compare binding patterns before and after cytokine stimulation

• Cytokine stimulation assays:

  • Treat cells with Stat5-activating cytokines (e.g., IL-2, IL-7, GM-CSF)

  • Monitor NCAM2 expression changes via RT-qPCR and Western blot

  • Perform time-course experiments to determine optimal stimulation conditions

  • Include Stat5 inhibitor controls to confirm specificity

• Reporter gene assays:

  • Clone the NCAM2 intronic region containing Stat5 binding sites into a reporter construct

  • Transfect cells and measure reporter activity following cytokine stimulation

  • Introduce point mutations in the Stat5 binding site to confirm specificity

• Flow cytometric analysis:

  • Stimulate cells with appropriate cytokines

  • Stain with FITC-conjugated NCAM2 antibody (bs-11094R-FITC) at 1:20-1:100 dilution

  • Analyze NCAM2 surface expression changes

  • Correlate with intracellular Stat5 phosphorylation status

This experimental approach would provide insights into the cytokine-dependent regulation of NCAM2 and potentially reveal new therapeutic targets for conditions where NCAM2 dysregulation is implicated.

How can NCAM2 antibodies be used to investigate the role of NCAM2 in neurite outgrowth and FGFR signaling?

NCAM2 has been shown to interact with FGFR and induce neurite outgrowth through activation of the Ras-MAPK pathway . To investigate this phenomenon:

• Neurite outgrowth assays:

  • Culture primary neurons or neuronal cell lines on substrate-coated plates

  • Add recombinant NCAM2 FnIII domains at varying concentrations

  • Treat cultures with NCAM2 antibodies (blocking or non-blocking)

  • Visualize neurites with immunostaining for β-III-tubulin or MAP2

  • Quantify neurite length, branching, and number using image analysis software

• FGFR activation studies:

  • Stimulate cells with recombinant NCAM2 FnIII domains

  • Assess FGFR phosphorylation by immunoprecipitation and Western blotting

  • Monitor downstream activation of Ras-MAPK pathway components (phospho-ERK1/2)

  • Use FITC-conjugated NCAM2 antibodies to visualize NCAM2-FGFR co-localization by confocal microscopy

• Perturbation experiments:

  • Apply NCAM2-derived peptides known to induce neurite outgrowth

  • Use NCAM2 antibodies to block specific domains and assess functional consequences

  • Compare effects of NCAM1 versus NCAM2 perturbation

  • Employ FGFR inhibitors to confirm pathway specificity

• Live-cell imaging:

  • Transfect neurons with fluorescent protein-tagged NCAM2 constructs

  • Use fluorescence resonance energy transfer (FRET) to assess NCAM2-FGFR interactions

  • Monitor growth cone dynamics in real-time following antibody application

  • Correlate spatial distribution of NCAM2 with neurite extension patterns

These approaches would help elucidate the mechanistic details of how NCAM2 contributes to neurite outgrowth through FGFR-dependent signaling and how this differs from NCAM1-mediated effects.

What are the optimal storage conditions for maintaining FITC-conjugated NCAM2 antibody activity?

To maintain optimal activity of FITC-conjugated NCAM2 antibodies:

• Storage temperature: Store at -20°C in a non-frost-free freezer to prevent temperature fluctuations .

• Aliquoting: Upon receipt, divide the antibody into small single-use aliquots to minimize freeze-thaw cycles. Repeated freeze-thaw cycles can lead to protein denaturation and fluorophore degradation .

• Buffer conditions: The antibody is typically supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol . This formulation helps maintain stability during storage.

• Light protection: FITC is susceptible to photobleaching. Store in amber vials or wrap containers in aluminum foil to protect from light exposure.

• Working dilution handling: Once diluted for use, keep the working solution on ice and protected from light. Use within the same day for optimal results.

• Stability monitoring: Periodically test antibody activity using a standard sample. A decrease in signal intensity may indicate degradation.

Following these guidelines can significantly extend the shelf-life and maintain the performance of FITC-conjugated NCAM2 antibodies.

How can I validate NCAM2 antibody specificity for my specific application?

Validating antibody specificity is crucial for reliable experimental results. For NCAM2 antibodies:

• Positive and negative controls:

  • Positive tissue controls: Use brain tissue (high NCAM2 expression)

  • Negative tissue controls: Use tissues with minimal NCAM2 expression

  • Recombinant protein: Test with purified recombinant human NCAM2 protein

• Knockdown/knockout validation:

  • Perform siRNA knockdown of NCAM2

  • Use CRISPR/Cas9-mediated knockout models

  • Compare antibody staining between wild-type and NCAM2-deficient samples

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (amino acids 51-150 of human NCAM2 for bs-11094R-FITC)

  • Apply to duplicate samples

  • Loss of signal indicates specificity for the target epitope

• Orthogonal detection methods:

  • Confirm results using antibodies targeting different NCAM2 epitopes

  • Correlate protein detection with mRNA expression (RT-qPCR)

  • Compare results across multiple applications (e.g., IHC, WB, flow cytometry)

• Cross-reactivity assessment:

  • Test reactivity with related proteins (e.g., NCAM1)

  • If cross-reactivity exists, determine if it affects your specific application

Western blot data has validated the specificity of NCAM2 antibodies by detecting the predicted 93 kDa band in both pig and rat brain lysates, as well as in recombinant human NCAM2 protein samples .

What are the considerations for developing dual immunofluorescence protocols incorporating FITC-conjugated NCAM2 antibodies?

Developing dual or multiple immunofluorescence protocols requires careful planning:

• Fluorophore selection:

  • Choose secondary fluorophores with minimal spectral overlap with FITC (excitation ~495 nm, emission ~520 nm)

  • Recommended combinations: FITC + Cy3/Rhodamine or FITC + Cy5/Alexa Fluor 647

• Primary antibody compatibility:

  • When combining with other rabbit antibodies: Use sequential staining protocols with intermediate blocking steps

  • For antibodies from different species: Simultaneous incubation is possible

  • Verify that epitope accessibility is not affected by dual staining

• Optimization steps:

  • Titrate each antibody individually before combining

  • Test order of antibody application (sequential vs. simultaneous)

  • Optimize blocking to minimize background

  • Include single-stained controls for spectral bleed-through assessment

• Analysis considerations:

  • Include proper controls for autofluorescence and spectral unmixing

  • For co-localization studies, include appropriate statistical analyses

  • Consider super-resolution techniques for detailed co-localization studies

• Protocol example for NCAM2/FGFR co-localization:

  Step	Procedure	Time	Temperature
  1	Fix cells/tissue (4% PFA)	10-15 min	RT
  2	Permeabilize (0.1% Triton X-100)	10 min	RT
  3	Block (5% normal serum)	1 hour	RT
  4	Incubate with FITC-NCAM2 antibody (1:50)	2 hours	RT or overnight
  5	Wash 3x with PBS	5 min each	RT
  6	Incubate with anti-FGFR antibody	2 hours	RT or overnight
  7	Wash 3x with PBS	5 min each	RT
  8	Incubate with secondary antibody for FGFR	1 hour	RT
  9	Wash 3x with PBS	5 min each	RT
  10	Counterstain nuclei (DAPI)	5 min	RT
  11	Mount and image	-	-

This protocol can be adapted based on specific experimental requirements and tissue types.

How can I design experiments to compare NCAM1 versus NCAM2 function using specific antibodies?

Designing comparative studies of NCAM1 and NCAM2 requires careful experimental planning:

• Expression profiling:

  • Use parallel immunohistochemistry with specific antibodies for NCAM1 and NCAM2

  • Perform Western blot analysis of multiple tissues/cell types

  • Quantify relative expression levels by flow cytometry

• Functional domain comparison:

  • Design experiments targeting the FnIII domains of both proteins

  • Compare ATP binding capabilities using NCAM1 and NCAM2 FnIII2 domains

  • Assess FGFR binding affinity and activation kinetics

• Neurite outgrowth assays:

  • Treat neuronal cultures with recombinant NCAM1 or NCAM2 FnIII domains

  • Compare concentration-response relationships

  • Use domain-specific blocking antibodies to identify functional epitopes

  • Assess downstream signaling activation (Ras-MAPK pathway)

• Binding partner identification:

  • Perform immunoprecipitation with specific antibodies

  • Analyze co-precipitated proteins by mass spectrometry

  • Validate interactions with co-localization studies using fluorescent antibodies

• Loss-of-function studies:

  • Generate specific knockdowns/knockouts for NCAM1 or NCAM2

  • Perform rescue experiments with domain swap constructs

  • Assess phenotypic consequences in relevant cellular contexts

This experimental design would highlight the unique properties of NCAM2 compared to NCAM1, particularly regarding their roles in neurite outgrowth and FGFR signaling.

What are the methodological approaches for investigating NCAM2 in tissue-specific contexts?

Investigating NCAM2 in tissue-specific contexts requires tailored methodological approaches:

• Tissue-specific expression analysis:

  • Perform immunohistochemistry using anti-NCAM2 antibodies on multiple tissue types

  • Use paraffin-embedded sections with optimized antigen retrieval protocols

  • Compare NCAM2 localization patterns across tissues

  • Document concentration requirements (e.g., 20 μg/ml has been effective for human kidney tissue)

• Cell type identification in heterogeneous tissues:

  • Develop dual immunofluorescence protocols with cell type-specific markers

  • Use FITC-conjugated NCAM2 antibodies (bs-11094R-FITC) at 1:20-1:100 dilution

  • Perform confocal microscopy for co-localization analysis

  • Consider laser capture microdissection for isolating NCAM2-positive cells

• Tissue-specific function assessment:

  • Develop conditional knockout models targeting NCAM2 in specific tissues

  • Use tissue-specific promoters to drive Cre recombinase expression

  • Analyze phenotypic consequences of tissue-restricted NCAM2 deletion

  • Perform rescue experiments with wild-type or mutant NCAM2

• Developmental regulation:

  • Compare NCAM2 expression across developmental stages

  • Analyze temporal regulation in specific tissues

  • Investigate the role of Stat5 in tissue-specific NCAM2 regulation

  • Create developmental timelines of NCAM2 expression and function

These approaches would provide insights into the tissue-specific roles of NCAM2 and potentially reveal new therapeutic targets for conditions where NCAM2 dysregulation is implicated in specific tissues.

How can NCAM2 antibodies be utilized in emerging single-cell analysis techniques?

NCAM2 antibodies, particularly FITC-conjugated variants, can be integrated into cutting-edge single-cell analysis platforms:

• Single-cell RNA-seq paired with protein detection:

  • Use FITC-conjugated NCAM2 antibodies in CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing)

  • Sort NCAM2-positive cells by FACS prior to single-cell RNA-seq

  • Correlate NCAM2 protein levels with transcriptomic profiles

  • Identify co-expression patterns with other neural adhesion molecules

• Mass cytometry (CyTOF):

  • Conjugate NCAM2 antibodies with rare earth metals

  • Integrate into multi-parameter panels (30+ markers)

  • Perform dimensional reduction analyses to identify NCAM2-associated cell populations

  • Map NCAM2 expression onto developmental trajectories

• Spatial transcriptomics:

  • Combine FITC-NCAM2 immunofluorescence with spatial transcriptomics platforms

  • Map NCAM2 protein distribution relative to mRNA expression patterns

  • Integrate with multiplexed error-robust FISH (MERFISH) for subcellular resolution

  • Develop computational workflows for integrating protein and RNA spatial data

• Super-resolution microscopy:

  • Apply STORM/PALM techniques with NCAM2 antibodies

  • Investigate nanoscale organization of NCAM2 at the cell membrane

  • Study co-clustering with FGFR and other binding partners

  • Analyze dynamics using live-cell super-resolution approaches

These emerging techniques will provide unprecedented insights into NCAM2 biology at single-cell and subcellular resolution, potentially revealing heterogeneity in expression and function that conventional bulk approaches cannot detect.

What are the considerations for using NCAM2 antibodies in studies of neurodevelopmental disorders?

NCAM2 has been implicated in neurodevelopmental disorders, particularly Down syndrome, due to its role in neural development . When designing studies:

• Patient sample considerations:

  • Optimize protocols for fixed post-mortem tissue

  • Develop protocols for induced pluripotent stem cell (iPSC)-derived neurons

  • Consider bioethical implications and consent requirements

  • Include appropriate age and sex-matched controls

• Genotype-phenotype correlations:

  • Analyze NCAM2 expression in relation to genetic variants

  • Use NCAM2 antibodies to quantify protein levels in different genetic backgrounds

  • Correlate NCAM2 expression with severity of neurological phenotypes

  • Develop flow cytometry panels including NCAM2-FITC for patient-derived cells

• Therapeutic development applications:

  • Screen for compounds that normalize aberrant NCAM2 expression

  • Use NCAM2 antibodies to validate target engagement

  • Develop assays for high-throughput screening

  • Consider antibody-based therapeutic approaches (targeting or mimicking NCAM2)

• Model system selection:

  • Validate NCAM2 antibody cross-reactivity with model organisms

  • Develop appropriate transgenic models (e.g., trisomy 21 mouse models)

  • Consider organoid systems for 3D neural development studies

  • Use CRISPR/Cas9 to create isogenic iPSC lines with NCAM2 variants

These approaches would advance our understanding of NCAM2's role in neurodevelopmental disorders and potentially identify new therapeutic strategies. The slight overexpression of NCAMs, which increases homotypic adhesion properties of cells, may contribute to Down syndrome phenotypes , making NCAM2 an important target for investigation.