SYNE2 Antibody

What is SYNE2/Nesprin-2 and what is its cellular function?

Nesprin-2 (SYNE2) is a nuclear envelope protein that serves as a critical linker between cytoskeletal components of the cytoplasm and the nucleoplasm. As a member of actin-binding proteins that localize to the outer nuclear membrane, Nesprin-2 plays a crucial role in maintaining nuclear architecture and cellular mechanics . The protein functions within the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, facilitating signal transduction between the cytoplasm and nucleus, and contributing to nuclear positioning, cell migration, and mechanotransduction pathways. Understanding this structural and functional role is essential for researchers designing experiments targeting nuclear envelope dynamics and associated pathologies.

What are the main isoforms of SYNE2 and how do they differ?

SYNE2 generates multiple protein isoforms through complex mechanisms of alternative splicing, alternative promoter usage, and alternative terminator usage. These isoforms vary significantly in both domain architecture and molecular weight:

The Nesprin-2 Giant isoform is particularly notable as it contains the complete domain structure, including the N-terminal actin-binding domain, multiple spectrin repeat (SR) domains, and the C-terminal KASH domain that anchors to the nuclear envelope . Smaller isoforms may lack specific domains, resulting in altered functional properties and localization patterns. For research applications, it is critical to determine which isoform(s) are being targeted by the selected antibody, as this will significantly impact experimental design and interpretation.

How should researchers select the appropriate SYNE2 antibody for their specific application?

Selecting the appropriate SYNE2 antibody requires careful consideration of multiple technical factors tailored to the specific experimental application:

• Target epitope location: Determine whether your research requires targeting specific domains of SYNE2. For example, antibodies targeting the spectrin repeat domains (like those recognizing residues 360-440) may be suitable for studying the full-length protein .

• Validated applications: Confirm the antibody has been validated for your specific application. Some antibodies are only validated for certain techniques - such as Boster's A02818 antibody which is guaranteed for IHC applications , while others like Proteintech's 25265-1-AP are validated for WB, IF, and ELISA .

• Species reactivity: Verify compatibility with your experimental model. For instance, antibody A02818 reacts with human and mouse SYNE2 , while 25265-1-AP shows reactivity with human, mouse, and rat samples .

• Clonality considerations: Polyclonal antibodies (like those in the search results) often provide robust signal detection across multiple epitopes but may show batch-to-batch variation. For applications requiring consistent epitope recognition, monoclonal antibodies might be preferable.

• Published validation data: Request or review validation data demonstrating the antibody's specificity and sensitivity in applications similar to your intended use.

The selection process should be guided by your specific research objectives and experimental design requirements rather than general availability or cost considerations.

What validation steps should be performed before using a new SYNE2 antibody?

Before incorporating a new SYNE2 antibody into your research protocol, implement these essential validation steps to ensure reliable and reproducible results:

• Positive and negative control testing: Use samples with known SYNE2 expression profiles (e.g., HEK-293 cells have been verified for some SYNE2 antibodies) alongside appropriate negative controls (SYNE2 knockdown cells or tissues from knockout models).

• Cross-reactivity assessment: Particularly important for polyclonal antibodies, evaluate potential cross-reactivity with related proteins, especially other nesprin family members.

• Molecular weight verification: Compare the observed molecular weight with expected values for specific isoforms. For example, while the calculated molecular weight of Nesprin-2 Giant is approximately 796-799 kDa, some antibodies detect smaller isoforms in the 50-60 kDa range .

• Reproducibility testing: Perform technical replicates across different experimental conditions to assess consistency of results.

• Orthogonal validation: When possible, confirm findings using alternative methods or antibodies targeting different epitopes of SYNE2.

• Dilution optimization: Perform titration experiments to determine optimal working concentrations. For example, for Proteintech's 25265-1-AP, recommended dilutions are 1:500-1:1000 for Western blot and 1:10-1:100 for immunofluorescence .

Thorough validation will significantly enhance the reliability and interpretability of subsequent experimental results.

What are the optimal conditions for using SYNE2 antibodies in immunohistochemistry (IHC)?

Optimizing IHC protocols for SYNE2 detection requires careful consideration of several technical parameters:

Researchers should document all optimization steps methodically to ensure reproducibility across experiments and facilitate troubleshooting if needed.

How can researchers effectively use SYNE2 antibodies in Western blotting applications?

Successful Western blotting for SYNE2 detection presents unique challenges due to its large molecular weight and multiple isoforms. Follow these methodological considerations:

• Sample preparation optimization:

  • Use strong lysis buffers containing ionic detergents (e.g., RIPA with 0.1% SDS)

  • Include protease inhibitors to prevent degradation

  • Sonicate samples briefly to shear DNA and reduce viscosity

• Gel electrophoresis considerations:

  • For Nesprin-2 Giant detection (~796 kDa), use low percentage (3-4%) acrylamide gels

  • Consider gradient gels (4-12%) to resolve multiple isoforms simultaneously

  • Extend running time at lower voltage to improve resolution of high molecular weight proteins

• Transfer optimization:

  • For large isoforms, employ wet transfer with extended transfer times (overnight at 30V)

  • Use lower percentage methanol (5-10%) in transfer buffer for high molecular weight proteins

  • Consider specialized transfer systems designed for large proteins

• Antibody parameters:

  • Dilute primary antibodies according to manufacturer recommendations (e.g., 1:500-1:1000 for Proteintech's 25265-1-AP)

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

  • Use appropriate blocking agents to minimize background

• Signal detection considerations:

  • Employ enhanced chemiluminescence (ECL) systems with extended exposure times

  • For quantitative analysis, consider fluorescence-based detection methods

• Data interpretation:

  • Verify molecular weights against expected isoform sizes

  • Note that some antibodies may preferentially detect specific isoforms (e.g., 50-60 kDa isoforms rather than the full-length 796 kDa protein)

These methodological refinements will help overcome the technical challenges associated with SYNE2 Western blotting.

How can SYNE2 antibodies be used to investigate neurodevelopmental disorders?

SYNE2 antibodies serve as critical tools for investigating the relationship between SYNE2 mutations and neurodevelopmental disorders, based on recent evidence linking biallelic SYNE2 mutations to intellectual disability and autism spectrum disorder . A comprehensive experimental approach would include:

• Patient-derived cell models:

  • Establish fibroblast or lymphoblastoid cell lines from patients with SYNE2 mutations

  • Use SYNE2 antibodies for immunoblotting to quantify protein expression levels, as demonstrated in studies revealing significant reduction of nesprin-2 giant protein in patient cells carrying mutations in the SR5 domain

  • Employ immunofluorescence to examine subcellular localization patterns and nuclear morphology

• Brain organoid systems:

  • Generate cerebral organoids from patient iPSCs or genome-edited stem cells

  • Apply immunofluorescence with SYNE2 antibodies to analyze protein expression and distribution during neurodevelopment

  • Proteintech's 25265-1-AP antibody has been validated for human dorsal cerebral organoid applications

• Animal model validation:

  • Characterize SYNE2 expression in mouse models carrying analogous mutations

  • Perform detailed immunohistochemistry across developmental timepoints

  • Correlate protein expression patterns with behavioral and cognitive phenotypes

• Mechanistic investigations:

  • Use co-immunoprecipitation with SYNE2 antibodies to identify altered protein interactions

  • Combine with chromatin immunoprecipitation to investigate potential effects on genome organization and gene expression

• Therapeutic screening platforms:

  • Employ SYNE2 antibodies as readouts in high-throughput screens for compounds that restore normal protein levels or localization

This multifaceted approach leverages SYNE2 antibodies as both analytical tools and potential therapeutic development instruments.

What techniques can be used to study SYNE2 conformational changes in disease states?

Investigating conformational changes in SYNE2 requires sophisticated methodological approaches that parallel those used for other proteins known to undergo structural alterations in pathological conditions, such as α-synuclein in Parkinson's disease :

• Conformation-specific antibody development:

  • Generate and characterize antibodies that preferentially recognize disease-associated conformations

  • This approach has been successfully employed for α-synuclein, where antibodies like Syn 505, Syn 506, and Syn 514 recognize conformational variants associated with pathological inclusions

  • For SYNE2, antibodies targeting specific spectrin repeat domains (such as SR5) where pathogenic mutations occur could be developed

• Biochemical fractionation coupled with immunoblotting:

  • Separate detergent-soluble and detergent-insoluble protein fractions

  • Use SYNE2 antibodies to detect shifts in solubility profiles that may indicate conformational changes

  • Similar approaches have revealed accumulation of α-syn in detergent-insoluble fractions from diseased brain

• Immunofluorescence microscopy with quantitative analysis:

  • Compare staining patterns between wild-type and mutant SYNE2 proteins

  • Implement blinded quantification methods to analyze immunofluorescence data

  • Statistical analysis techniques such as paired t-tests can be employed to evaluate differences

• Epitope mapping approaches:

  • Employ truncation mutants and synthetic peptides to determine antibody epitopes

  • Use ELISA methods with synthetic peptides corresponding to different regions of SYNE2

  • This approach has been successful in mapping epitopes for conformation-specific α-synuclein antibodies

• Biophysical techniques combined with immunodetection:

  • Apply circular dichroism or fluorescence spectroscopy to monitor structural changes

  • Validate with immunoprecipitation using conformation-specific antibodies

These methodologies provide complementary approaches to characterizing SYNE2 conformational changes in neurodevelopmental disorders and other pathological conditions.

What are the common challenges in detecting SYNE2 isoforms and how can they be addressed?

Detection of SYNE2 isoforms presents several technical challenges that can be systematically addressed through methodological refinements:

• High molecular weight detection limitations:

  • Challenge: The Nesprin-2 Giant isoform (~796-799 kDa) is often difficult to detect in standard Western blotting

  • Solution: Use specialized large-protein electrophoresis systems with gradient gels (3-8% Tris-Acetate), extended transfer times, and nitrocellulose membranes with larger pore sizes

• Multiple isoform interpretation:

  • Challenge: The presence of numerous isoforms (ranging from 30 kDa to 799 kDa) complicates data interpretation

  • Solution: Use isoform-specific antibodies when available; otherwise, carefully document all detected bands and compare with predicted molecular weights for known isoforms

• Epitope masking in fixed tissues:

  • Challenge: Nuclear envelope proteins may have reduced antibody accessibility

  • Solution: Optimize antigen retrieval methods specifically for nuclear envelope proteins; test multiple retrieval buffers and conditions

• Cross-reactivity with other nesprin family members:

  • Challenge: Sequence homology between SYNE1, SYNE2, and other family members

  • Solution: Validate antibody specificity using SYNE2 knockout or knockdown controls; consider using multiple antibodies targeting different epitopes

• Variable expression levels across tissues:

  • Challenge: SYNE2 expression varies significantly among tissue types

  • Solution: Optimize protein loading and detection sensitivity for each tissue type; use positive control samples with known expression levels

• Degradation during sample preparation:

  • Challenge: Large proteins are particularly susceptible to proteolytic degradation

  • Solution: Use fresh samples when possible; include multiple protease inhibitors in lysis buffers; maintain samples at 4°C throughout processing

Systematic optimization addressing these challenges will significantly improve detection sensitivity and specificity for SYNE2 isoforms.

How can researchers verify SYNE2 antibody specificity in their experimental systems?

Comprehensive verification of SYNE2 antibody specificity requires a multi-faceted approach integrating multiple validation strategies:

• Genetic knockdown/knockout validation:

  • Implement siRNA-mediated knockdown of SYNE2 in cell culture systems

  • Where available, utilize CRISPR/Cas9-engineered knockout cell lines or tissues from knockout animals

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

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide (when available)

  • For example, blocking peptides for antibodies like A02818 can be purchased based on the immunogen (synthetic peptide derived from human protein at AA range: 360-440)

  • Specific signal should be competitively inhibited while non-specific binding will remain

• Multiple antibody concordance:

  • Compare results using antibodies targeting different SYNE2 epitopes

  • Consistent localization and molecular weight detection across antibodies increases confidence in specificity

• Heterologous expression systems:

  • Overexpress tagged versions of SYNE2 (full-length or specific isoforms)

  • Confirm antibody detection correlates with expression levels

  • Compare detection of endogenous versus overexpressed protein

• Mass spectrometry validation:

  • Perform immunoprecipitation with the SYNE2 antibody followed by mass spectrometry analysis

  • Confirm the presence of SYNE2 peptides in the immunoprecipitated material

  • Analyze for potential cross-reactive proteins

• Cross-species reactivity assessment:

  • Test antibody performance across species with known sequence conservation or divergence

  • For example, confirm whether antibodies with claimed human and mouse reactivity show expected pattern of reactivity based on sequence homology

These rigorous validation steps ensure experimental results can be confidently attributed to specific detection of SYNE2 proteins.

How might SYNE2 antibodies contribute to understanding the molecular basis of neurodevelopmental disorders?

SYNE2 antibodies hold significant potential to advance our understanding of neurodevelopmental disorders through several innovative research directions:

• Mechanistic characterization of pathogenic variants:

  • Use antibodies to quantify the expression levels of nesprin-2 giant in patient-derived cells carrying different SYNE2 mutations

  • Building on findings that biallelic mutations in the SR5 domain lead to hypo-expression , investigate whether different mutation types affect protein levels through distinct mechanisms

  • Compare mutations in different domains to establish structure-function relationships

• Developmental expression profiling:

  • Apply SYNE2 antibodies to track temporal and spatial expression patterns during neurodevelopment

  • Utilize immunofluorescence in brain organoid models at different developmental stages

  • Correlate SYNE2 expression patterns with critical neurodevelopmental processes

• Nuclear-cytoskeletal coupling investigation:

  • Exploit SYNE2 antibodies in high-resolution microscopy to examine nuclear positioning defects

  • Investigate potential disruptions in mechanotransduction pathways in neurons with SYNE2 mutations

  • Analyze interactions with cytoskeletal components in the context of neuronal migration and positioning

• Protein-protein interaction networks:

  • Employ SYNE2 antibodies in proximity labeling approaches to map the interactome in wild-type versus mutant contexts

  • Identify potential compensatory mechanisms when SYNE2 function is compromised

  • Discover novel therapeutic targets within the SYNE2 interaction network

• Circuit-level consequences:

  • Combine SYNE2 immunostaining with electrophysiology to correlate protein expression with functional outcomes in neuronal circuits

  • Investigate whether specific neural populations are differentially affected by SYNE2 dysfunction

These research directions leverage the specificity and versatility of SYNE2 antibodies to bridge molecular defects with neurodevelopmental consequences, potentially identifying new therapeutic avenues for associated disorders.

What emerging techniques might enhance the utility of SYNE2 antibodies in research?

Emerging technologies offer promising opportunities to extend the experimental applications of SYNE2 antibodies:

• Super-resolution microscopy integration:

  • Apply techniques like STORM, PALM, or STED microscopy with SYNE2 antibodies to visualize nanoscale organization at the nuclear envelope

  • Investigate structural changes in the LINC complex in disease states with unprecedented resolution

  • Combine with multiplexed imaging to examine SYNE2 colocalization with interacting partners

• Single-cell proteomics applications:

  • Develop SYNE2 antibody-based approaches for analyzing protein expression at single-cell resolution

  • Integrate with spatial transcriptomics to correlate protein localization with gene expression patterns

  • Identify cell type-specific alterations in SYNE2 expression in complex tissues

• In vivo imaging adaptations:

  • Generate nanobody or scFv derivatives of SYNE2 antibodies suitable for intracellular expression

  • Develop fluorescent protein fusions for live-cell and in vivo imaging of SYNE2 dynamics

  • Apply these tools to monitor SYNE2 behavior during neurodevelopmental processes in real-time

• Therapeutic antibody engineering:

  • Explore intrabody approaches to modulate SYNE2 function or stability in disease contexts

  • Develop antibody-drug conjugates for targeted modulation of SYNE2-expressing cells

  • Design bispecific antibodies linking SYNE2 to potential compensatory proteins

• Cryo-electron tomography applications:

  • Use SYNE2 antibodies conjugated to electron-dense markers for precise localization

  • Investigate structural organization of the nuclear envelope and LINC complex components

  • Compare native versus disease-associated conformations at near-atomic resolution

These technological innovations promise to overcome current limitations in studying SYNE2 biology and pathology, potentially yielding transformative insights into nuclear envelope function in health and disease.