CXorf36 Antibody

What is CXorf36 and why is it relevant to neurodevelopmental research?

CXorf36, also known as Deleted in autism-related protein 1 (DIA1R) or Divergent protein kinase domain 2B (DIPK2B), is an X-linked gene that has significant associations with autism spectrum disorders (ASDs). The protein encoded by this gene is particularly relevant to neurodevelopmental research because mutations and variations in CXorf36 have been linked to X-linked autism susceptibility (AUTSX2). Studies indicate that CXorf36's expression patterns and functional roles may be critical in understanding the molecular mechanisms underlying autism pathophysiology.

The gene is strongly associated with several core ASD phenotypes, making antibodies against this protein valuable tools for investigating autism's molecular basis . Recent research suggests CXorf36 may be involved in cellular signaling pathways relevant to neurodevelopment, though its precise function remains under investigation.

For optimal preservation of antibody activity, researchers should follow these evidence-based handling practices:

• Store unconjugated CXorf36 antibodies at -20°C in their original formulation, typically containing glycerol to prevent freeze-thaw damage .

• For working solutions, store at 4°C for short-term use (1-2 weeks) and avoid repeated freeze-thaw cycles, which can cause protein denaturation and diminished antibody performance.

• Most commercial CXorf36 antibodies are supplied in buffered solutions (pH 7.4 PBS) with preservatives like sodium azide (0.05% NaN3) and stabilizers (40% Glycerol) .

• When transporting or shipping the antibody between laboratories, maintain cold chain conditions, preferably on wet ice for short transports or dry ice for longer shipments .

• Prior to use, centrifuge the antibody solution briefly to collect all liquid at the bottom of the vial and ensure homogeneity when pipetting.

Following these protocols will help maintain antibody specificity and sensitivity, particularly important for detecting potentially low-abundance proteins like CXorf36 in neurological samples.

What are the key considerations for optimizing Western blot protocols with CXorf36 antibodies?

When performing Western blot analysis with CXorf36 antibodies, researchers should consider several optimization strategies based on published validation data:

• Sample preparation: For optimal detection of CXorf36, use RIPA buffer supplemented with protease inhibitors for cell or tissue lysis.

• Gel percentage selection: Based on validation data, 8% SDS-PAGE gels provide appropriate resolution for CXorf36 detection .

• Primary antibody dilution: Start with the recommended 1:200 dilution for rabbit polyclonal antibodies against CXorf36, but titration may be necessary for optimal signal-to-noise ratio in your specific sample type .

• Secondary antibody selection: For unconjugated primary antibodies, goat anti-rabbit IgG conjugated to HRP at 1:8000 dilution has been validated to provide specific detection .

• Exposure time: Begin with 40 seconds exposure time as a baseline, then adjust based on signal intensity and background levels .

When troubleshooting weak signals, consider increasing protein loading (validated protocols used 40 μg of total protein from HeLa cells) or extending primary antibody incubation time to overnight at 4°C to enhance sensitivity while maintaining specificity.

How can researchers validate the specificity of CXorf36 antibodies in their experimental systems?

To ensure antibody specificity, which is critical for accurate data interpretation, researchers should implement the following validation approaches:

• Positive controls: Use cell lines with documented CXorf36 expression, such as HeLa cells, which have been validated for CXorf36 antibody testing .

• Blocking peptide competition: Perform parallel experiments with the antibody pre-incubated with the immunizing peptide (such as the synthetic peptide of human CXorf36 used for immunization) to confirm signal specificity.

• siRNA knockdown validation: Transfect cells with CXorf36-targeted siRNA and confirm reduction in antibody signal correlates with reduced expression.

• Multiple antibody comparison: When possible, validate findings using at least two different antibodies targeting distinct epitopes of CXorf36 to confirm consistent localization or expression patterns.

• Cross-reactivity testing: If working with non-human models, perform appropriate cross-reactivity tests as most available CXorf36 antibodies are primarily validated against human samples .

For immunohistochemistry applications specifically, include appropriate negative controls (omitting primary antibody) and consider using known positive tissue sections based on Human Protein Atlas data for CXorf36 expression patterns .

What are the typical expression patterns of CXorf36 in human tissues, and how should immunohistochemistry be optimized?

Based on immunohistochemistry data from the Human Protein Atlas project referenced in the antibody documentation, CXorf36 shows tissue-specific expression patterns that researchers should consider when designing experiments:

• Brain tissue expression: CXorf36 shows notable expression in various brain regions, consistent with its proposed role in neurodevelopmental disorders.

• Cellular localization: The protein has been detected primarily in neuronal cells, with both cytoplasmic and membrane-associated patterns described.

For optimal immunohistochemistry protocols with CXorf36 antibodies:

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended for formalin-fixed, paraffin-embedded (FFPE) tissues.

• Antibody dilution range: Begin with the validated range of 1:20-1:50 for IHC applications with rabbit polyclonal antibodies .

• Incubation conditions: Overnight incubation at 4°C can improve specific staining while reducing background.

• Detection system: For maximum sensitivity in detecting potentially low-abundance CXorf36, use polymer-based detection systems conjugated with HRP.

• Counterstaining: Hematoxylin counterstaining provides optimal contrast for visualizing CXorf36 immunoreactivity in tissue contexts.

Researchers should be aware that expression patterns may vary between developmental stages and disease states, particularly in conditions where CXorf36 has been implicated, such as autism spectrum disorders .

How do mutations or variations in CXorf36 correlate with specific autism phenotypes, and what methods are most effective for studying these relationships?

The relationship between CXorf36 variations and autism phenotypes represents an area of active research. Current data indicates:

To effectively study these relationships, researchers should employ:

• Integrated genomic-phenotypic analysis: Correlate specific CXorf36 variants (SNPs, CNVs, indels) with detailed phenotypic data using comprehensive statistical models that account for additional genetic and environmental factors.

• Functional assays in neuronal models: Utilize CRISPR/Cas9 gene editing to introduce specific CXorf36 variants into neuronal cell lines or organoids, then assess changes in neuronal morphology, electrophysiological properties, and protein interaction networks.

• Animal models: Develop and characterize transgenic mouse models expressing human CXorf36 variants to evaluate behavioral, cellular, and molecular phenotypes relevant to autism.

• Antibody-based tissue profiling: Apply validated CXorf36 antibodies in comparative immunohistochemistry studies of post-mortem brain tissue from individuals with and without autism, focusing on regions implicated in social cognition and behavior .

These approaches can help elucidate how CXorf36 variations contribute to the heterogeneous presentation of autism spectrum disorders and potentially identify targets for therapeutic intervention.

What are the challenges in differentiating between closely related family members when using CXorf36 antibodies, and how can these be addressed?

Researchers working with CXorf36 antibodies face several challenges related to specificity and cross-reactivity with related protein family members. These challenges and their solutions include:

• Epitope conservation issues: CXorf36 shares sequence homology with related protein family members, particularly with DIA1 (Deleted In Autism 1, also known as CDIPT2C). To address this:

  • Select antibodies raised against unique, non-conserved regions of CXorf36

  • Perform comprehensive epitope mapping to confirm specificity

  • Use computational analysis to identify potential cross-reactive proteins based on the immunogen sequence

• Validation in knockout systems: For definitive specificity validation:

  • Implement CRISPR/Cas9 knockout of CXorf36 in relevant cell lines

  • Compare antibody reactivity in wild-type versus knockout samples across multiple applications

  • Document any residual signal that might indicate cross-reactivity

• Parallel detection methods: To confirm antibody-based findings:

  • Complement immunodetection with mRNA expression analysis (RT-qPCR, in situ hybridization)

  • Use mass spectrometry-based proteomics for antibody-independent protein identification

  • Consider proximity ligation assays for enhanced specificity in tissue samples

• Pre-adsorption controls: For critical experiments:

  • Pre-incubate the antibody with recombinant proteins of related family members

  • Compare signal patterns to identify potentially confounding cross-reactivity

  • Include gradient concentrations of blocking peptides to establish specificity thresholds

These methodological approaches are particularly important when studying CXorf36 in the context of neurodevelopmental disorders, where precise protein identification is crucial for understanding disease mechanisms and developing potential therapeutic strategies.

How can researchers effectively use CXorf36 antibodies in conjunction with other molecular tools to investigate its role in cellular signaling pathways potentially disrupted in autism?

To comprehensively investigate CXorf36's role in cellular signaling pathways relevant to autism, researchers should integrate antibody-based detection with complementary molecular approaches:

• Proximity-based protein interaction studies:

  • Implement BioID or APEX2 proximity labeling with CXorf36 as the bait protein

  • Use co-immunoprecipitation with validated CXorf36 antibodies followed by mass spectrometry

  • Apply proximity ligation assays in fixed cells or tissues to detect native protein interactions

• Subcellular localization dynamics:

  • Combine immunofluorescence using CXorf36 antibodies with markers for specific cellular compartments

  • Track potential translocation events following cellular stimulation relevant to neuronal signaling

  • Implement live-cell imaging with CXorf36-fluorescent protein fusions to complement antibody-based static imaging

• Phosphoproteomic analysis:

  • Use phospho-specific antibodies (if available) or mass spectrometry to identify CXorf36 phosphorylation states

  • Map kinase-substrate relationships using kinase inhibitors and CXorf36 antibodies

  • Correlate phosphorylation changes with functional readouts in neuronal models

• Transcriptional network analysis:

  • Perform ChIP-seq or CUT&RUN using CXorf36 antibodies if nuclear localization is observed

  • Integrate with RNA-seq data from CXorf36 knockdown/overexpression models

  • Map downstream gene expression changes related to autism-associated pathways

• High-content screening approaches:

  • Utilize automated imaging with CXorf36 antibodies to screen compound libraries

  • Identify modulators of CXorf36 expression, localization, or post-translational modifications

  • Correlate phenotypic changes with alterations in CXorf36 status

By combining these approaches, researchers can build a systems-level understanding of CXorf36's functional role in neurodevelopmental processes and potentially identify points of therapeutic intervention for autism spectrum disorders .

What are the common sources of background or non-specific staining when using CXorf36 antibodies, and how can these be mitigated?

Researchers frequently encounter background issues when working with CXorf36 antibodies. The following strategies address specific sources of non-specific staining:

• Endogenous peroxidase activity (for HRP-based detection):

  • Implement a dedicated peroxidase quenching step (3% H₂O₂ in methanol for 10-15 minutes) prior to primary antibody incubation

  • For neuronal tissues, which can have high endogenous peroxidase activity, extend quenching time or use alternative detection systems

• Non-specific antibody binding:

  • Optimize blocking conditions using 5% normal serum from the same species as the secondary antibody

  • Include 0.1-0.3% Triton X-100 in blocking solutions for balanced permeabilization

  • Consider adding 1% BSA to reduce hydrophobic interactions

  • Use commercial antibody diluents specifically formulated to reduce background

• Cross-reactivity with related proteins:

  • Increase antibody dilution beyond standard recommendations (test ranges from 1:100 to 1:500)

  • Pre-absorb the antibody with recombinant related proteins

  • Consider using monoclonal antibodies if available, which typically offer higher specificity than polyclonals

• Tissue fixation artifacts:

  • Optimize fixation protocols (duration, fixative concentration)

  • Implement antigen retrieval methods tailored to CXorf36 detection (citrate buffer pH 6.0 has been validated)

  • For difficult samples, consider alternative fixatives or light fixation protocols

• Autofluorescence (for fluorescent detection):

  • Treat sections with Sudan Black B (0.1% in 70% ethanol) to reduce lipofuscin autofluorescence

  • Use spectral imaging and linear unmixing to separate specific signal from autofluorescence

  • Consider longer wavelength fluorophores to avoid tissue autofluorescence regions

Carefully titrating both primary and secondary antibodies while maintaining consistent washing protocols (recommended: 3x5 minutes in PBS-T) can significantly improve signal-to-noise ratio when working with CXorf36 antibodies .

How can researchers effectively generate and validate custom CXorf36 antibodies for specialized applications?

For researchers requiring specialized CXorf36 antibodies beyond commercially available options, the following comprehensive approach is recommended:

• Strategic epitope selection:

  • Target unique regions of CXorf36 with low homology to related proteins

  • Focus on regions with predicted surface exposure using structural prediction tools

  • Consider multiple epitopes (N-terminal, C-terminal, and internal) for complementary detection

  • Analyze conservation across species if cross-reactivity is desired

• Immunization and production strategies:

  • For maximum specificity, use synthetic peptides conjugated to carrier proteins (KLH or BSA)

  • Consider recombinant protein fragments for broader epitope recognition

  • Implement ELISA-based screening against both immunizing antigen and full-length protein

  • Purify antibodies using affinity chromatography with the immunizing peptide

• Rigorous validation pipeline:

  • Establish specificity using Western blot against recombinant CXorf36 and cell lysates

  • Confirm detection of endogenous protein in positive control samples (e.g., HeLa cells)

  • Validate absence of signal in knockout or knockdown models

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

  • Cross-validate with commercially available antibodies targeting different epitopes

• Application-specific optimization:

  • For each intended application (WB, IHC, IF, IP, etc.), determine optimal working dilutions

  • Document fixation and sample preparation requirements

  • Establish reproducible protocols with detailed methodology

  • Create validation data packages with representative images and controls

This systematic approach ensures the generation of reliable research tools for advancing CXorf36 research, particularly important given its potential significance in neurodevelopmental disorders .

What are the best practices for multiplexing CXorf36 antibodies with other neuronal markers in brain tissue analysis?

Multiplexed immunostaining enables comprehensive analysis of CXorf36 in the context of specific cell types and subcellular compartments. The following best practices optimize multiplexed detection:

• Antibody selection criteria for compatibility:

  • Choose primary antibodies raised in different host species to avoid cross-reactivity

  • When using multiple rabbit antibodies (including CXorf36 antibodies), implement sequential staining with intervening blocking steps

  • Verify each antibody individually before attempting multiplexing

  • Consider using directly conjugated primary antibodies to simplify protocols

• Optimized multiplexing protocols:

  • For conventional immunofluorescence:

    • Apply tyramide signal amplification (TSA) for detection of low-abundance targets

    • Use species-specific secondary antibodies with minimal cross-reactivity

    • Select fluorophores with well-separated excitation/emission spectra

  • For chromogenic multiplexing:

    • Implement sequential detection with intervening stripping or blocking

    • Use distinctly colored chromogens with good contrast (e.g., DAB, Fast Red, Vector Blue)

• Recommended marker combinations with CXorf36:

  • Neuronal subtype markers: MAP2 (dendrites), NeuN (neuronal nuclei), TH (dopaminergic neurons)

  • Glial markers: GFAP (astrocytes), IBA1 (microglia), MBP (oligodendrocytes)

  • Subcellular compartment markers: GM130 (Golgi), LAMP1 (lysosomes), Synaptophysin (synaptic vesicles)

• Advanced multiplexing technologies:

  • Consider cyclic immunofluorescence methods for higher-order multiplexing

  • Implement clearing techniques (CLARITY, CUBIC) for thick-section or whole-organ imaging

  • Apply spectral imaging with linear unmixing to resolve overlapping fluorophores

  • Utilize image registration for correlating serial sections

• Quantification and analysis approaches:

  • Implement automated cell segmentation for population-level analyses

  • Quantify colocalization using established metrics (Pearson's, Manders' coefficients)

  • Apply spatial statistics to characterize distribution patterns

  • Consider deep learning approaches for complex pattern recognition

These approaches enable researchers to place CXorf36 in its appropriate cellular context, particularly important when investigating its potential role in autism spectrum disorders and other neurodevelopmental conditions .

How might single-cell analysis techniques using CXorf36 antibodies advance our understanding of cell-type specific expression in neurodevelopmental disorders?

Single-cell analysis represents a frontier in understanding the cell-type specific relevance of CXorf36 in neurodevelopmental disorders. Methodological approaches include:

• Single-cell immunoprofiling strategies:

  • Apply multiplexed ion beam imaging (MIBI) or imaging mass cytometry (IMC) incorporating CXorf36 antibodies to map expression at single-cell resolution

  • Implement CyTOF with CXorf36 antibodies conjugated to rare earth metals for high-dimensional phenotyping

  • Utilize sequential immunofluorescence methods to expand the number of proteins that can be analyzed alongside CXorf36

• Integration with single-cell transcriptomics:

  • Combine immunofluorescence using CXorf36 antibodies with laser capture microdissection and subsequent scRNA-seq

  • Implement CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) approaches with oligo-tagged CXorf36 antibodies

  • Correlate protein expression with transcriptional signatures at single-cell resolution

• Spatial transcriptomics approaches:

  • Use spatial transcriptomics methods to map CXorf36 mRNA expression in tissue contexts

  • Validate with immunofluorescence using CXorf36 antibodies on serial sections

  • Develop computational approaches to integrate spatial protein and mRNA data

• Developmental trajectory analysis:

  • Apply CXorf36 antibodies across developmental timepoints to map expression changes

  • Correlate with emerging cellular phenotypes and circuit formation

  • Implement lineage tracing in combination with CXorf36 immunodetection

These approaches would significantly advance understanding of CXorf36's role in specific neural cell populations and potentially identify cell types particularly vulnerable in autism spectrum disorders. This could provide crucial insights for targeted therapeutic development and improved understanding of autism pathophysiology .

What role might post-translational modifications of CXorf36 play in its function, and how can researchers investigate these modifications?

Post-translational modifications (PTMs) potentially regulate CXorf36 function, representing an unexplored dimension of its biology relevant to neurodevelopmental disorders. Research strategies include:

• Computational prediction and mapping:

  • Apply PTM prediction algorithms to identify potential modification sites (phosphorylation, glycosylation, ubiquitination)

  • Map these sites to protein domains to predict functional consequences

  • Generate structural models incorporating predicted PTMs

• Mass spectrometry-based PTM profiling:

  • Immunoprecipitate CXorf36 using validated antibodies followed by MS/MS analysis

  • Implement enrichment strategies for specific modifications (phosphopeptide enrichment, glycopeptide enrichment)

  • Compare PTM profiles between normal and disease-relevant conditions

• PTM-specific antibody development:

  • Generate modification-specific antibodies (e.g., phospho-CXorf36) based on MS findings

  • Validate these antibodies using in vitro modified recombinant proteins

  • Apply in cellular and tissue contexts to map modification dynamics

• Functional consequences of PTMs:

  • Implement site-directed mutagenesis to generate modification-mimetic or modification-deficient CXorf36 variants

  • Assess effects on protein-protein interactions, subcellular localization, and stability

  • Evaluate downstream signaling consequences in neuronal models

• Enzyme identification:

  • Conduct screens to identify kinases, phosphatases, or other modifying enzymes acting on CXorf36

  • Validate enzyme-substrate relationships using in vitro and cellular assays

  • Investigate these enzymes as potential therapeutic targets

Given the association of CXorf36 with autism spectrum disorders, understanding its post-translational regulation could reveal critical mechanisms underlying neurodevelopmental pathology and potentially identify novel intervention points .

How can researchers utilize CXorf36 antibodies in patient-derived models to advance personalized medicine approaches for autism spectrum disorders?

Patient-derived models represent a powerful approach for translating CXorf36 research into personalized medicine strategies for autism spectrum disorders. Methodological frameworks include:

• Patient-derived cellular models:

  • Generate induced pluripotent stem cells (iPSCs) from individuals with CXorf36 variants

  • Differentiate into neural progenitors and mature neuronal subtypes

  • Apply CXorf36 antibodies to track protein expression, localization, and interaction patterns

  • Compare with isogenic corrected lines to isolate variant-specific effects

• Brain organoid applications:

  • Develop 3D cerebral organoids from patient-derived iPSCs

  • Implement immunohistochemistry with CXorf36 antibodies to assess expression patterns

  • Apply live imaging approaches to track developmental trajectories

  • Compare structural and functional phenotypes between patient and control organoids

• High-throughput screening platforms:

  • Establish reporter systems based on CXorf36 function in patient-derived neural cells

  • Screen compound libraries for modulators of aberrant phenotypes

  • Utilize automated imaging with CXorf36 antibodies as readouts for therapeutic efficacy

  • Develop patient-specific intervention strategies based on screening results

• Biomarker development:

  • Evaluate CXorf36 protein levels or modifications in accessible patient samples (e.g., blood-derived neuronal organoids)

  • Correlate with clinical phenotypes and treatment responses

  • Develop standardized immunoassays suitable for clinical application

  • Implement longitudinal monitoring during developmental trajectories and interventions

These approaches can bridge fundamental CXorf36 research with clinical applications, potentially enabling stratification of autism spectrum disorder patients based on molecular mechanisms and development of targeted therapeutic strategies. The combination of patient-derived models with validated CXorf36 antibodies provides a powerful platform for advancing precision medicine in neurodevelopmental disorders .