NCAM2 Antibody

What is NCAM2 and what is its role in the nervous system?

NCAM2 (neural cell adhesion molecule 2) is one of two neural cell adhesion molecules encoded in the mammalian genome, the other being NCAM1. As an adhesion molecule with brain-enriched expression patterns, NCAM2 serves critical functions in neuronal development and maintenance .

NCAM2 is primarily involved in:

• Axonal targeting and guidance

• Neurite branching processes

• Synaptic development and maturation

• Cell-cell adhesion in neural tissues

NCAM2 has been genetically linked to Down syndrome and may play important roles in neurodegenerative conditions such as Alzheimer's disease . Its expression is most robust in both adult and fetal brain tissues, suggesting sustained importance throughout neural development and into maturity .

What applications are NCAM2 antibodies commonly used for?

NCAM2 antibodies are versatile research tools applicable across multiple experimental platforms. Based on manufacturer specifications, these antibodies demonstrate utility in:

The optimal dilution should be determined experimentally for each application and specific antibody, as performance can vary based on tissue type, fixation method, and experimental conditions .

What molecular weight should be expected when detecting NCAM2?

When using NCAM2 antibodies in Western blot applications, researchers should expect to observe specific molecular weight bands reflecting the protein's native state and potential post-translational modifications:

The presence of multiple bands reflects glycosylation states and potential isoforms of NCAM2. Researchers should be aware that differences in tissue types, sample preparation methods, and gel running conditions may affect the apparent molecular weight observed in experiments .

What species reactivity can be expected from commercial NCAM2 antibodies?

Current commercial NCAM2 antibodies demonstrate cross-reactivity across several mammalian species:

When studying species not listed as confirmed, researchers should perform validation experiments to ensure antibody cross-reactivity before proceeding with full-scale studies .

What are the recommended storage conditions for NCAM2 antibodies?

Proper storage is critical for maintaining antibody functionality and specificity. Based on manufacturer recommendations:

• Store at -20°C for long-term stability

• Most formulations remain stable for one year after shipment when stored properly

• Antibodies are typically supplied in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting is generally unnecessary for -20°C storage

• Some manufacturers specifically indicate not to aliquot the antibody

Some antibody preparations may contain 0.1% BSA in smaller volume formats (e.g., 20μl sizes) , which should be noted when designing experiments sensitive to bovine proteins.

How can I optimize NCAM2 antibody dilutions for different experimental applications?

Optimizing antibody dilutions is crucial for balancing signal strength against background. Different applications require distinct optimization approaches:

For Western Blotting:

• Begin with manufacturer's recommended range (e.g., 1:1000 for Cell Signaling #64422, 1:5000-1:50000 for Proteintech antibodies)

• Perform a dilution series experiment (e.g., 1:1000, 1:2000, 1:5000, 1:10000)

• Include positive controls (brain tissue lysates show strong expression)

• Optimal dilution provides clear specific bands at 93-140 kDa with minimal background

For Immunofluorescence:

• Start with recommended dilution range (1:50-1:500)

• Test fixation methods (paraformaldehyde typically works well for neural tissues)

• Include antigen retrieval optimization if working with fixed tissues

• Monitor signal-to-noise ratio across dilutions

For Immunohistochemistry:

• Begin testing with mid-range dilution (e.g., 1:200)

• For NCAM2, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 provides an alternative approach

• Validate specificity using known positive tissues (brain samples) and negative controls

For each application, tissue-dependent optimization is essential as NCAM2 expression levels vary significantly across neural and non-neural tissues .

What antigen retrieval methods are recommended for NCAM2 immunohistochemistry?

Effective antigen retrieval is particularly important for detecting NCAM2 in fixed tissue sections:

• Primary recommendation: TE buffer at pH 9.0 for heat-induced epitope retrieval

• Alternative method: Citrate buffer at pH 6.0

• Heat-induced retrieval methods (pressure cooker, microwave, or water bath) are generally more effective than enzymatic methods

• Optimal retrieval time should be empirically determined (typically 10-20 minutes at 95-100°C)

• For formalin-fixed, paraffin-embedded (FFPE) tissues, more aggressive retrieval may be necessary compared to frozen sections

When optimizing antigen retrieval for NCAM2, researchers should monitor both signal intensity and tissue morphology preservation, as excessive retrieval can damage tissue integrity while insufficient retrieval may result in false negatives .

How do I troubleshoot multiple bands or unexpected molecular weights when using NCAM2 antibodies?

The presence of multiple bands or unexpected molecular weights in Western blot analysis of NCAM2 requires systematic troubleshooting:

Normal pattern interpretation:

• NCAM2 typically appears at 93 kDa (calculated molecular weight) and 125-140 kDa (glycosylated forms)

• The presence of both bands is expected and reflects normal post-translational modification

Troubleshooting unexpected patterns:

• Additional higher molecular weight bands (>140 kDa): May indicate aggregation; add fresh reducing agents or heat samples more thoroughly

• Additional lower molecular weight bands (<93 kDa): May indicate degradation; improve sample preparation with additional protease inhibitors

• No bands or very weak signal: Consider sample source (brain tissues have higher expression than other tissues)

• Smeared bands: May indicate excessive protein loading or incomplete protein denaturation

To confirm band specificity:

• Use different NCAM2 antibodies targeting distinct epitopes

• Include positive control lysates from brain tissues with known NCAM2 expression

• Consider peptide competition assays to confirm specificity

• For ambiguous results, consider IP-Western approaches to enrich for NCAM2 prior to detection

How does NCAM2 antibody performance differ between monoclonal and polyclonal options?

The choice between monoclonal and polyclonal NCAM2 antibodies involves important experimental considerations:

Monoclonal NCAM2 antibodies (e.g., Proteintech #68205-1-Ig):

• Provide consistent lot-to-lot reproducibility

• Recognize a single epitope, potentially reducing background

• Typically show higher specificity but may be more sensitive to epitope masking

• Useful for applications requiring high specificity (e.g., immunofluorescence)

• Mouse IgG2b isotype (for Proteintech #68205-1-Ig)

Polyclonal NCAM2 antibodies (e.g., Proteintech #13850-1-AP, Affinity Biosciences #DF4224):

• Recognize multiple epitopes on the NCAM2 protein

• Often provide stronger signals due to binding multiple sites

• Less affected by minor changes in protein conformation or epitope masking

• Useful for applications requiring sensitive detection (e.g., low abundance proteins)

• Typically rabbit-derived for commercial offerings

Application-specific considerations:

• For Western blotting, both types perform well, with polyclonals often providing stronger signals

• For IP applications, polyclonal antibodies often perform better due to recognizing multiple epitopes

• For IHC/IF, monoclonals may provide cleaner background but potentially at the cost of signal intensity

Researchers should select antibody format based on their specific experimental needs, considering factors such as required specificity, signal strength, and application type .

What are the considerations for using NCAM2 antibodies in studying neurodegenerative diseases?

NCAM2 has been implicated in neurodegenerative conditions, particularly Alzheimer's disease, making antibody selection crucial for studying these disorders:

Experimental design considerations:

• Select antibodies validated in disease-relevant models or tissues

• For Alzheimer's disease studies, antibodies should detect both normal and pathologically altered NCAM2

• Consider epitope accessibility in protein aggregates common to neurodegenerative conditions

• Include age-matched controls when studying NCAM2 in aged tissues

Methodological approaches:

• Co-localization studies with disease markers (e.g., Aβ plaques, tau tangles)

• Sequential extraction protocols to examine NCAM2 in soluble versus insoluble fractions

• Quantitative Western blotting to assess potential changes in NCAM2 processing

• Examination of potential NCAM2 fragments that might be disease-specific

Research relevance:

• NCAM2 is genetically linked to Down syndrome, which shows early-onset Alzheimer's pathology

• NCAM2 functions in axonal targeting and synaptic development, processes disrupted in multiple neurodegenerative conditions

• Changes in NCAM2 glycosylation or proteolytic processing may serve as biomarkers for disease progression

When designing studies of NCAM2 in neurodegenerative contexts, researchers should carefully consider tissue preparation methods that preserve disease-specific protein modifications while enabling effective antibody recognition .

How can I effectively use NCAM2 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) with NCAM2 antibodies requires careful optimization to preserve protein-protein interactions while achieving specific enrichment:

Recommended protocol:

• Use polyclonal antibodies (e.g., Proteintech #13850-1-AP) which are more effective for IP applications

• Recommended antibody amount: 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate

• Use mild lysis buffers (e.g., NP-40 or CHAPS-based) to preserve protein complexes

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Perform IP at 4°C overnight with gentle rotation

• Include appropriate negative controls (non-specific IgG of same species/isotype)

Tissue considerations:

• Brain tissue lysates show strongest NCAM2 expression and best IP results

• Fresh tissue preparation provides better results than frozen samples

• Include protease inhibitors to prevent NCAM2 degradation

• Consider crosslinking approaches for capturing transient interactions

Validation methods:

• Confirm successful IP by Western blotting a small portion of the IP product

• For Co-IP studies, validate interactions using reciprocal IP when possible

• Consider mass spectrometry analysis of IP products to identify novel interactors

The Proteintech #13850-1-AP antibody has been validated for IP applications using mouse brain tissue, making it a reliable choice for NCAM2 interaction studies .

What buffer systems optimize NCAM2 antibody performance in various applications?

Buffer composition significantly impacts NCAM2 antibody performance across different applications:

For Western blotting:

• Sample preparation: RIPA buffer with protease inhibitors effectively extracts NCAM2

• Transfer buffer: Standard Towbin buffer (25 mM Tris, 192 mM glycine) with 20% methanol

• Blocking: 5% non-fat milk in TBST provides good results with minimal background

• Antibody dilution: Primary antibodies are best diluted in 5% BSA in TBST to reduce background

• Recommended dilutions range from 1:1000 to 1:50000 depending on the specific antibody

For immunohistochemistry:

• Antigen retrieval: TE buffer at pH 9.0 (primary recommendation) or citrate buffer at pH 6.0 (alternative)

• Blocking: 10% normal serum (species different from primary antibody source)

• Antibody dilution: 1:50-1:500 in antibody diluent containing 1% BSA in PBS-T

• Washing: TBS-T (0.1% Tween-20)

For immunofluorescence:

• Fixation: 4% paraformaldehyde in PBS for 15-20 minutes

• Permeabilization: 0.1-0.3% Triton X-100 in PBS for 10 minutes

• Blocking: 5-10% normal serum with 1% BSA in PBS

• Antibody dilution: 1:50-1:500 in 1% BSA in PBS-T

For immunoprecipitation:

• Lysis buffer: Non-denaturing buffers (NP-40 or CHAPS-based) with protease inhibitors

• Binding buffer: PBS or TBS with 0.1% Tween-20

• Wash buffer: Increasing stringency washes (TBS-T with gradually increasing salt concentration)

Optimal buffer conditions should be determined empirically for each specific application and tissue type .

What tissue preparation protocols yield optimal results with NCAM2 antibodies?

Proper tissue preparation is critical for successful NCAM2 detection, particularly given its enrichment in brain tissues:

For Western blotting samples:

• Fresh tissue homogenization in RIPA buffer with protease inhibitor cocktail

• Brain tissues (particularly cerebellum) yield strongest NCAM2 signal

• Recommended protein loading: 20-50 μg total protein per lane

• Include sample reducing agent and heat to 95°C for 5 minutes before loading

For immunohistochemistry:

• Fixation: 10% neutral-buffered formalin (24-48 hours)

• Processing: Standard paraffin embedding protocol

• Section thickness: 4-6 μm sections optimal

• Antigen retrieval: TE buffer pH 9.0 (primary) or citrate buffer pH 6.0 (alternative)

• Validated in mouse brain and human gliomas tissues

For immunofluorescence:

• Fresh-frozen sections: 10-14 μm optimal thickness

• Fixation: 4% paraformaldehyde (10-15 minutes) or ice-cold methanol (10 minutes)

• Permeabilization: 0.2% Triton X-100 in PBS (10 minutes)

• Validated in mouse brain tissues

For cell cultures:

• Fixation: 4% paraformaldehyde (15 minutes at room temperature)

• Permeabilization: 0.1% Triton X-100 (5-10 minutes)

• Neural cell lines or primary cultures show better NCAM2 expression than non-neural lines

Researchers should note that NCAM2 detection is most robust in neural tissues, with cerebellum and brain samples showing particularly strong expression .

How do I select the right NCAM2 antibody clone for specific research questions?

Selecting the appropriate NCAM2 antibody requires matching antibody characteristics to specific research needs:

For basic expression studies:

• Both monoclonal and polyclonal options work well

• Choose based on application (e.g., Proteintech #68205-1-Ig for WB and IF-P; #13850-1-AP for WB, IHC, and IP)

• Consider species reactivity requirements based on your experimental model

For mechanistic studies of NCAM2 function:

• Select antibodies whose epitopes don't interfere with functional domains

• Consider function-blocking antibodies if studying adhesion properties

• For studies of NCAM2 interactions, use antibodies validated for IP applications

For disease-related studies:

• Choose antibodies validated in disease-relevant tissues

• Consider epitope location relative to disease-associated modifications

• For neurodegenerative disease studies, select antibodies that recognize both glycosylated forms (93kDa and 125-140kDa)

For detection method compatibility:

• Western blotting: Most NCAM2 antibodies perform well

• IHC/IF: Consider background issues based on tissue type; monoclonals may give cleaner results

• Flow cytometry: Select antibodies specifically validated for this application

• Super-resolution microscopy: Higher affinity antibodies generally perform better

When possible, validate key findings with multiple NCAM2 antibodies targeting different epitopes to ensure result reproducibility and specificity .

What controls should be used when validating NCAM2 antibody specificity?

Rigorous validation of NCAM2 antibody specificity requires appropriate controls:

Positive controls:

• Brain tissue lysates (human, mouse, or rat) for Western blotting

• Brain tissue sections (particularly cerebellum) for IHC/IF

• Cell lines with confirmed NCAM2 expression (e.g., LNCaP)

• Recombinant NCAM2 protein (for antibody validation)

Negative controls:

• Tissues with minimal NCAM2 expression (non-neural tissues)

• Primary antibody omission controls

• Isotype-matched irrelevant antibody controls

• Peptide competition/blocking experiments

Specificity validation approaches:

• siRNA/shRNA knockdown of NCAM2 (should reduce/eliminate signal)

• CRISPR/Cas9 knockout models (complete elimination of specific signal)

• Comparison across multiple NCAM2 antibodies targeting different epitopes

• Western blot analysis to confirm expected molecular weight patterns (93kDa and 125-140kDa)

Cross-reactivity assessment:

• Test for potential cross-reactivity with NCAM1 (the closest homolog)

• Validate specificity in tissues expressing both NCAM1 and NCAM2

• Consider peptide array analysis to verify epitope specificity

Thorough validation using multiple complementary approaches increases confidence in antibody specificity and experimental results .

How do I address cross-reactivity concerns with NCAM1 when using NCAM2 antibodies?

NCAM1 and NCAM2 share structural similarities that can potentially lead to antibody cross-reactivity issues:

Preventative approaches:

• Select antibodies specifically raised against unique NCAM2 epitopes

• Review the immunogen information provided by manufacturers

• The immunogen sequence can be verified against NCAM1 using protein BLAST to assess potential cross-reactivity

• Commercial antibodies are generally validated for NCAM2 specificity

Analytical validation:

• Run parallel Western blots with purified NCAM1 and NCAM2 proteins

• Include tissues with differential expression of NCAM1 vs. NCAM2

• Brain tissues express both proteins, while certain peripheral tissues express predominantly NCAM1

Experimental controls:

• Include NCAM2 knockdown/knockout samples to confirm specificity

• For critical experiments, consider peptide competition assays with both NCAM1 and NCAM2 peptides

• Pre-absorption controls with recombinant proteins can distinguish specific from cross-reactive binding

Interpretation considerations:

• NCAM1 and NCAM2 have different molecular weight profiles in Western blots

• NCAM1 typically shows bands at 120-180 kDa (several isoforms)

• NCAM2 shows distinct bands at 93 kDa and 125-140 kDa

By implementing these approaches, researchers can minimize concerns about NCAM1/NCAM2 cross-reactivity in their experiments.

What strategies help resolve inconsistent staining patterns in NCAM2 immunofluorescence?

Inconsistent immunofluorescence staining can significantly impact NCAM2 research. Several strategies can help resolve these issues:

Sample preparation optimization:

• Standardize fixation protocols (duration, temperature, fixative composition)

• For NCAM2, 4% paraformaldehyde for 15-20 minutes typically works well

• Optimize permeabilization (0.1-0.3% Triton X-100 for 10 minutes)

• Use freshly prepared buffers and reagents

Antibody-related adjustments:

• Titrate antibody concentration carefully (1:50-1:500 recommended range)

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

• Consider different antibody clones if persistent issues occur

• Verify antibody stability and storage conditions

Signal detection optimization:

• Implement signal amplification methods for weak signals

• Adjust exposure settings consistently across experiments

• Use appropriate filters to minimize autofluorescence

• Consider spectral unmixing for tissues with high autofluorescence

Protocol modifications for difficult samples:

• For highly fixed tissues, increase antigen retrieval strength

• For frozen sections, optimize section thickness (10-14 μm typically optimal)

• For cultured cells, adjust confluence and culture conditions

• Consider alternative secondary antibodies if background is problematic

Mouse brain tissue has been validated for NCAM2 immunofluorescence using Proteintech #68205-1-Ig, providing a reliable positive control for protocol optimization .

How can I quantitatively analyze NCAM2 expression in comparative studies?

Quantitative analysis of NCAM2 expression requires careful methodological considerations:

For Western blot quantification:

• Use internal loading controls (β-actin, GAPDH, or β-tubulin)

• Include a standard curve with known protein amounts

• Consider both the 93 kDa and higher molecular weight bands (125-140 kDa) in analysis

• Use fluorescent secondary antibodies for wider linear detection range

• Normalize to total protein loading (using stain-free gels or total protein stains)

For immunohistochemistry quantification:

• Standardize all staining parameters (time, temperature, reagent concentrations)

• Process all samples in parallel to minimize batch effects

• Use automated image analysis software to reduce subjective bias

• Establish clear criteria for positive staining

• Include region-matched controls for brain tissue analysis

For immunofluorescence quantification:

• Collect images with identical acquisition parameters

• Use appropriate controls for background subtraction

• Employ colocalization analysis for spatial relationship studies

• Consider z-stack imaging for three-dimensional analysis

• Apply consistent thresholding across all experimental groups

Statistical approaches:

• Perform power analysis to determine appropriate sample sizes

• Use appropriate statistical tests based on data distribution

• Consider both parametric and non-parametric approaches

• Account for multiple comparisons when analyzing different brain regions

Consistent methodology across experimental groups is critical for reliable quantitative comparison of NCAM2 expression levels .

How do post-translational modifications of NCAM2 affect antibody recognition?

Post-translational modifications (PTMs) significantly impact NCAM2 antibody recognition and experimental interpretation:

Glycosylation effects:

• NCAM2 exists in multiple glycosylated forms (93 kDa non-glycosylated, 125-140 kDa glycosylated)

• Different antibodies may preferentially recognize specific glycosylation states

• Western blot typically shows both forms, with varying intensity ratios

• Brain region-specific glycosylation patterns may affect staining intensity

Other potential PTMs affecting recognition:

• Phosphorylation states (potentially affecting epitope accessibility)

• Proteolytic processing (generating fragments with altered antibody reactivity)

• Conformational changes induced by protein-protein interactions

Experimental approaches to address PTM variability:

• Use multiple antibodies targeting different epitopes

• Employ enzymatic deglycosylation (PNGase F treatment) to normalize glycosylation

• Compare reducing vs. non-reducing conditions for disulfide-dependent epitopes

• For disease studies, consider potential pathology-specific modifications

Documentation considerations:

• Clearly document which molecular weight forms were analyzed

• Note potential differences in PTM patterns across experimental conditions

• Consider tissue-specific differences in NCAM2 modification patterns

Researchers should be aware that the observed molecular weight pattern (93 kDa and 125-140 kDa bands) reflects normal NCAM2 biology rather than antibody non-specificity .

What tissue-specific considerations apply when using NCAM2 antibodies?

NCAM2 expression varies across tissues, necessitating tissue-specific experimental adaptations:

Brain tissue considerations:

• Highest NCAM2 expression levels, making it ideal for positive controls

• Cerebellum often shows particularly strong expression

• Requires careful handling to prevent protein degradation (rapid post-mortem processing)

• May require optimization of antigen retrieval for fixed samples

• Consider region-specific expression patterns in analysis

Human vs. rodent tissue differences:

• All major commercial antibodies show cross-reactivity with human, mouse, and rat NCAM2

• Expression patterns may vary between species

• Human tissue often requires more aggressive antigen retrieval

• Fixation artifacts may be more pronounced in human samples

• Human gliomas tissue has been validated for IHC applications

Cell line considerations:

• Neural-derived cell lines show higher expression than non-neural lines

• LNCaP cells have been validated for Western blot applications

• Primary neuronal cultures typically show strong NCAM2 expression

• Expression may be differentiation-state dependent

Disease-state tissue:

• Neurodegenerative conditions may alter NCAM2 processing or localization

• Down syndrome tissues may show altered expression patterns

• Consider altered cellular composition in disease states when interpreting results

• Compare only similarly processed samples for quantitative analysis

By adapting protocols to these tissue-specific considerations, researchers can optimize NCAM2 antibody performance across diverse experimental systems .