NLGN4Y Antibody

What is NLGN4Y and why is it significant for neurodevelopmental research?

NLGN4Y (Neuroligin 4, Y-linked) is a cell adhesion molecule located on the Y-chromosome that forms part of an X-Y gene pair with NLGN4X. This protein plays a crucial role in synaptic function and is primarily expressed at the postsynaptic side of synapses. NLGN4Y has been implicated in autism spectrum disorder (ASD) and intellectual disability, with particular significance in understanding the sex bias in ASD (affecting four times as many males as females) .

NLGN4Y is expressed in fetal and adult brain, as well as prostate, testis, and pancreas. As a synapse-associated protein, it is essential for the formation of functional synapses and may influence neuronal connectivity patterns . Research on NLGN4Y is particularly valuable for understanding sex-specific neurodevelopmental processes and the molecular basis of certain neurological conditions.

What types of NLGN4Y antibodies are currently available for research applications?

Several types of NLGN4Y antibodies are available for research:

• Monoclonal antibodies: Mouse-derived monoclonal antibodies like the 2F7 clone that offer high specificity .

• Polyclonal antibodies: Rabbit-derived polyclonal antibodies that may recognize multiple epitopes of NLGN4Y .

• Domain-specific antibodies: Antibodies targeting specific regions, such as the N-terminal region of NLGN4Y .

• Matched antibody pairs: Sets that include both capture and detection antibodies optimized for quantitative assays:

  • Capture Antibody: Mouse Monoclonal Anti-NLGN4Y (100 μg)

  • Detection Antibody: Rabbit MaxPab® affinity purified Polyclonal Anti-NLGN4Y (50 μg)

• Isoform-specific antibodies: Some antibodies can distinguish between different isoforms of NLGN4Y (isoform 1 and isoform 2) .

How should I select the appropriate NLGN4Y antibody for specific applications?

Selection of the appropriate NLGN4Y antibody depends on several experimental considerations:

When selecting an antibody, consider:

• Species reactivity: Ensure the antibody reacts with your target species (human, mouse, rat) .

• Application validation: Verify the antibody has been validated for your specific application .

• Cross-reactivity: Note that some NLGN4Y antibodies may cross-react with NLGN4X due to sequence similarity. This should be considered depending on your research question .

• Epitope location: Select antibodies targeting relevant domains based on your research goals .

What are the recommended protocols for optimizing Western blot analysis with NLGN4Y antibodies?

For optimal Western blot results with NLGN4Y antibodies:

• Sample preparation:

  • Use fresh tissue/cell lysates when possible

  • Include protease inhibitors to prevent degradation

  • For brain tissue, consider specialized extraction buffers for membrane proteins

• Gel electrophoresis:

  • Use 8-10% gels for optimal separation (NLGN4Y is approximately 92-100 kDa)

  • Load appropriate positive controls (e.g., A549 cells, HeLa cells, PC-3 cells, U-251 cells have shown positive results)

• Transfer and blocking:

  • For large proteins like NLGN4Y, use wet transfer with appropriate buffer

  • Block with 5% non-fat milk or BSA in TBST/PBST (optimize based on specific antibody)

• Antibody incubation:

  • Follow recommended dilution ranges (typically 1:500-1:2000 for most NLGN4Y antibodies)

  • Incubate primary antibody overnight at 4°C for optimal binding

  • Use appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG for polyclonal antibodies)

• Detection and analysis:

  • Expected molecular weight is approximately 92-100 kDa

  • Note that the mature form may be observed at 72 kDa

  • Use appropriate exposure times to avoid over-saturation

What controls are essential when working with NLGN4Y antibodies?

Proper controls are critical for interpreting results with NLGN4Y antibodies:

• Positive controls:

  • Cell lines with confirmed NLGN4Y expression (A549, HeLa, PC-3, U-251)

  • Brain tissue from appropriate species

  • Testis tissue (shows high expression)

  • Recombinant NLGN4Y protein (for antibody validation)

• Negative controls:

  • Samples from gene knockout models (where available)

  • Female-derived samples (for Y-linked specificity testing)

  • GST tag alone for antibodies raised against GST-tagged recombinant proteins

• Specificity controls:

  • Pre-absorption with immunizing peptide to confirm specificity

  • Secondary antibody only (omit primary antibody)

  • Isotype control antibody

• Cross-reactivity assessment:

  • Testing with NLGN4X expressing samples to assess potential cross-reactivity

  • Comparison with antibodies specific to NLGN4X

How can NLGN4Y antibodies be utilized to investigate autism spectrum disorders?

NLGN4Y antibodies offer several approaches for investigating autism spectrum disorders:

• Expression pattern analysis:

  • Compare NLGN4Y protein levels in post-mortem brain tissue from individuals with ASD versus controls

  • Investigate regional differences in expression across brain structures implicated in ASD

  • Examine developmental expression patterns during critical neurodevelopmental windows

• Synaptic localization studies:

  • Use immunofluorescence with NLGN4Y antibodies to visualize synaptic localization

  • Employ co-localization studies with other synaptic markers to assess synaptic integrity

  • Evaluate changes in synaptic distribution in ASD models

• Protein-protein interaction analysis:

  • Use co-immunoprecipitation with NLGN4Y antibodies to identify binding partners

  • Investigate interactions with neurexins and other synaptic proteins

  • Assess how ASD-associated mutations affect these interactions

• Functional studies:

  • Combine NLGN4Y antibody detection with electrophysiological recordings to correlate protein levels with synaptic function

  • Use antibodies to track NLGN4Y in trafficking studies in neuronal cultures

  • Apply antibodies in proximity ligation assays to examine molecular complexes at synapses

• Sex-specific neurobiological investigations:

  • Use NLGN4Y antibodies to explore the male-specific neural mechanisms potentially contributing to the 4:1 male:female ratio in ASD

  • Compare with NLGN4X expression patterns to understand sex chromosome gene dosage effects

How do maternal antibodies against NLGN4Y relate to neurodevelopment research?

Research has revealed intriguing connections between maternal antibodies to NLGN4Y and neurodevelopment:

• Maternal immune hypothesis:

  • Studies have found an association between maternal immune response to NLGN4Y and sexual orientation in male offspring

  • This suggests that maternal antibodies may cross the placenta and potentially influence fetal brain development

• Methodological approaches:

  • ELISA-based detection of anti-NLGN4Y antibodies in maternal plasma samples

  • Recombinant protein generation for NLGN4Y isoforms as antigens

  • Analysis of antibody levels against different NLGN4Y isoforms (isoform 1 and isoform 2)

• Research considerations:

  • Women have significantly higher anti-NLGN4Y levels than men (as expected for Y-linked proteins)

  • Mothers of sons with older brothers showed particularly elevated anti-NLGN4Y levels

  • The magnitude of maternal immune response appears to correlate with birth order of male offspring

• Mechanistic hypothesis:

  • Maternal antibodies to NLGN4Y may bind to and alter male-specific cell-surface molecules

  • This potentially affects masculinization of sex-dimorphic brain structures during development

  • NLGN4Y interactions with neurexins in synapse formation may influence brain development related to sexual/romantic attraction

How can I differentiate between NLGN4Y and its X-linked homolog NLGN4X in experimental systems?

Differentiating between NLGN4Y and NLGN4X requires careful experimental design:

• Antibody selection:

  • Use highly specific antibodies that do not cross-react between NLGN4Y and NLGN4X

  • Note that some antibodies may recognize both proteins due to sequence similarity

  • Validate antibody specificity using overexpression systems or sex-specific samples

• Genetic approaches:

  • Design PCR primers specific to unique regions of NLGN4Y and NLGN4X

  • Use Y-chromosome specific markers as controls

  • Consider RNA-seq for transcriptome-level distinction

• Experimental design:

  • Include male and female samples to leverage natural NLGN4Y absence in females

  • Use cell lines with known sex chromosome composition

  • Consider using XY and XX iPSC lines for controlled comparisons

• Protein analysis:

  • NLGN4Y and NLGN4X may have slightly different molecular weights that can be distinguished on high-resolution Western blots

  • 2D gel electrophoresis may separate the proteins based on charge differences

  • Mass spectrometry can identify unique peptides specific to each protein

• Immunoprecipitation approach:

  • Use IP with isoform-specific antibodies followed by mass spectrometry

  • Sequential IP can be used to deplete one isoform before capturing the other

What are common challenges when detecting NLGN4Y in tissue samples and how can they be overcome?

Researchers frequently encounter these challenges when detecting NLGN4Y:

• Low endogenous expression levels:

  • Use signal amplification methods (TSA, HRP polymers)

  • Optimize antibody concentration and incubation time

  • Consider more sensitive detection systems for Western blots

  • Use tissues with known high expression (brain, testis) as positive controls

• Cross-reactivity issues:

  • Validate antibody specificity using peptide competition assays

  • Include appropriate negative controls (female tissue for Y-linked specificity)

  • Consider using multiple antibodies targeting different epitopes

  • Pre-absorb antibodies if cross-reactivity is detected

• Protein degradation:

  • Use fresh samples when possible

  • Include protease inhibitor cocktails in extraction buffers

  • Optimize extraction conditions for membrane proteins

  • Store samples at appropriate temperatures (-20°C or -80°C) and avoid freeze/thaw cycles

• Fixation artifacts in IHC:

  • Optimize fixation conditions (type, duration, temperature)

  • Test different antigen retrieval methods

  • Consider using unfixed frozen sections if epitope is sensitive to fixation

  • Try different blocking reagents to reduce background

• Membrane protein solubilization:

  • Use appropriate detergents for membrane protein extraction

  • Consider specialized extraction buffers for synaptic proteins

  • Optimize sonication or homogenization protocols

How can I validate the specificity of my NLGN4Y antibody?

Rigorous validation of NLGN4Y antibodies ensures reliable experimental results:

• Western blot validation:

  • Run positive controls (recombinant protein, tissues with known expression)

  • Verify the expected molecular weight (approximately 92-100 kDa)

  • Check for absence of signal in female samples (Y-chromosome specificity)

  • Perform peptide competition assays with the immunizing peptide

• Immunohistochemistry validation:

  • Compare staining pattern with published literature

  • Perform dual labeling with established synaptic markers

  • Include appropriate negative controls

  • Compare results across multiple antibodies targeting different epitopes

• Knockout/knockdown validation:

  • Test antibody in NLGN4Y knockout models or CRISPR-edited cell lines

  • Use siRNA/shRNA knockdown to confirm signal reduction correlating with protein reduction

  • Test in overexpression systems to confirm increased signal

• Cross-reactivity assessment:

  • Test against recombinant NLGN4X protein

  • Compare results in paired male/female samples

  • Perform IP-MS to identify all proteins recognized by the antibody

• Batch testing:

  • Test each new lot of antibody against a standard sample

  • Maintain consistent positive controls across experiments

  • Document validation results for each lot

How should I interpret variations in NLGN4Y expression across different experimental conditions?

Interpreting NLGN4Y expression data requires careful consideration of several factors:

• Quantification methods:

  • Use appropriate normalization controls (housekeeping proteins, total protein stains)

  • Employ quantitative techniques (qWestern, ELISA) when possible

  • Consider the dynamic range of detection methods

  • Use independent methods to confirm significant changes

• Biological context:

  • Consider developmental stages (expression changes during neurodevelopment)

  • Account for brain region-specific expression patterns

  • Recognize cell type heterogeneity in tissue samples

  • Consider potential effects of neuronal activity on expression

• Technical considerations:

  • Evaluate the impact of sample preparation methods

  • Account for antibody affinity differences between batches

  • Control for potential post-translational modifications affecting detection

  • Consider the impact of protein localization changes versus expression changes

• Statistical analysis:

  • Use appropriate statistical tests for your experimental design

  • Consider biological versus technical replicates

  • Account for potential outliers

  • Perform power analysis to ensure adequate sample size

• Research applications:

  • When studying autism models, consider that subtle changes in NLGN4Y levels may have functional significance

  • In maternal antibody studies, normalize for potentially confounding variables (e.g., number of pregnancies)

  • For developmental studies, establish baseline expression curves before interpreting experimental changes

What are emerging applications of NLGN4Y antibodies in neurodevelopmental research?

Cutting-edge applications of NLGN4Y antibodies include:

• Single-cell protein analysis:

  • Using NLGN4Y antibodies in mass cytometry (CyTOF) for single-cell protein quantification

  • Combining with other markers to identify cell-specific expression patterns

  • Integrating with single-cell transcriptomics for multi-omics characterization

• Live imaging applications:

  • Developing non-perturbing antibody fragments for live neuron imaging

  • Using antibodies to track NLGN4Y trafficking in developing synapses

  • Super-resolution microscopy applications to visualize synaptic nanodomains

• Therapeutic targeting research:

  • Using antibodies to screen for compounds that modulate NLGN4Y function

  • Developing antibody-based approaches to normalize NLGN4Y activity in disease models

  • Target validation for neurodevelopmental disorders with NLGN4Y involvement

• Maternal-fetal interaction studies:

  • Further exploring the maternal immune hypothesis in neurodevelopmental conditions

  • Developing more sensitive assays for maternal anti-NLGN4Y antibodies

  • Investigating potential therapeutic approaches to mitigate maternal antibody effects

• Comparative studies across species:

  • Using antibodies to compare NLGN4Y localization and function across species with Y-linked neuroligins

  • Exploring evolutionary aspects of Y-chromosome neuroligins

How might NLGN4Y antibodies contribute to understanding sex differences in neurodevelopmental disorders?

NLGN4Y antibodies provide valuable tools for investigating sex-biased neurodevelopmental conditions:

• Male bias in autism research:

  • Investigating potential roles of NLGN4Y in the 4:1 male:female ratio observed in ASD

  • Comparing NLGN4Y and NLGN4X expression and function in male versus female brain development

  • Examining how sex chromosome genes interact with autosomal risk genes

• Developmental expression mapping:

  • Using NLGN4Y antibodies to map expression during critical neurodevelopmental windows

  • Comparing with NLGN4X expression timelines

  • Identifying sensitive periods when NLGN4Y function may be particularly important

• Maternal immune hypothesis investigations:

  • Further characterizing maternal antibody responses to NLGN4Y

  • Exploring potential mechanisms by which maternal antibodies might influence fetal brain development

  • Developing animal models to test causative relationships

• Synaptic function studies:

  • Examining potential sex differences in synaptic organization related to NLGN4Y

  • Investigating compensatory mechanisms in males with NLGN4Y dysfunction

  • Exploring synergistic effects with other sex-biased neuronal proteins

• Therapeutic implications:

  • Using NLGN4Y antibodies to evaluate sex-specific responses to interventions

  • Developing screening approaches for maternal antibodies as potential biomarkers

  • Exploring targeted approaches for normalizing NLGN4Y function in males with neurodevelopmental conditions