NSE4A Antibody

What is NSE4A and why is it relevant for molecular research?

NSE4A (Non-SMC Element 4 Homolog A, also known as NSMCE4A or C10orf86) is a critical component of the SMC5-SMC6 complex involved in DNA double-strand break repair through homologous recombination. This protein plays essential roles in genome stability, sister chromatid recombination, and telomere maintenance, particularly in alternative lengthening of telomeres (ALT) cell lines. NSE4A functions as a δ-kleisin component that mediates the sumoylation of shelterin complex components, which is proposed to lead to shelterin complex disassembly in ALT-associated PML bodies (APBs) . The protein's involvement in DNA damage response pathways makes it a valuable target for studies focused on genome integrity, DNA repair mechanisms, and telomere biology.

What are the key specifications of NSE4A antibodies for research applications?

NSE4A antibodies are available in various formats with specific characteristics optimized for different experimental applications. Typical specifications include:

These specifications are critical for experimental design and result interpretation when working with NSE4A antibodies in research contexts .

What are the optimal protocols for NSE4A antibody application in Western blotting?

For optimal Western blotting results with NSE4A antibodies, researchers should follow these methodological guidelines:

• Sample preparation: Extract nuclear proteins using specialized nuclear extraction buffers as NSE4A is primarily localized in the nucleus and at chromosomes/telomeres .

• Separation parameters: Use appropriate percentage SDS-PAGE gels (10-12%) that effectively resolve proteins in the 44kDa range (the calculated molecular weight of NSE4A) .

• Transfer and blocking: After electrophoresis, transfer proteins to PVDF or nitrocellulose membranes and block with 3-5% BSA in PBS-T buffer to minimize background.

• Antibody incubation: Dilute the NSE4A antibody 1:1000 in blocking buffer and incubate overnight at 4°C for optimal binding .

• Detection and visualization: Use appropriate secondary antibodies (anti-rabbit IgG) conjugated to HRP or fluorescent tags, depending on your detection system.

For validation purposes, include proper positive controls (tissues/cells known to express NSE4A) and negative controls (tissues/cells with NSE4A knockdown or from species not recognized by the antibody).

How can researchers validate the specificity of NSE4A antibodies?

Validating antibody specificity is crucial for reliable experimental results. For NSE4A antibodies, multiple complementary approaches should be employed:

• Competitive ELISA: Coat plates with recombinant NSE4A protein (approximately 46 ng/100 μl) and perform competitive binding assays with varying concentrations of soluble NSE4A antigen. Decreased signal with increasing antigen concentration confirms specific binding .

• Antigen competition in immunolabeling: Pre-incubate the NSE4A antibody with purified antigen (approximately 800 nM) before application to samples. Significant signal reduction compared to controls indicates specificity .

• Western blot validation: Confirm a single band at the expected molecular weight (approximately 44 kDa) in tissues known to express NSE4A.

• Genetic validation: Compare antibody staining patterns between wild-type samples and those from NSE4A knockout or knockdown models. The signal should be reduced or absent in the latter .

• Orthogonal validation: Verify results using alternative detection methods such as RNA-seq or mass spectrometry.

These validation steps ensure that experimental observations genuinely reflect NSE4A biology rather than non-specific interactions .

How can NSE4A antibodies be effectively used to study chromatin organization?

NSE4A antibodies provide valuable tools for investigating chromatin organization through several sophisticated approaches:

• Super-resolution microscopy: Utilizing structured illumination microscopy with NSE4A antibodies can reveal its euchromatin-specific localization in somatic interphase nuclei. This approach has demonstrated that NSE4A is absent from heterochromatin (DAPI-intense chromocenters) and nucleoli in Arabidopsis thaliana .

• Co-immunolabeling experiments: Combine NSE4A antibody labeling with markers for specific chromatin domains to map its precise nuclear distribution. In plant studies, NSE4A has been shown to colocalize with euchromatin markers but is excluded from heterochromatic regions .

• Chromatin immunoprecipitation (ChIP): NSE4A antibodies can be used in ChIP experiments to identify genomic regions where NSE4A binds, providing insights into its role in chromatin organization and DNA repair.

• Cell-cycle dependent analysis: Analyzing NSE4A localization across different cell cycle stages reveals dynamic patterns of association with chromatin. Research has shown that NSE4A operates in a cell cycle-dependent manner with changing localization patterns during interphase versus mitosis .

These techniques collectively enable detailed mapping of NSE4A's role in nuclear architecture and chromatin dynamics.

What approaches can resolve contradictory results when using NSE4A antibodies across different model systems?

When faced with contradictory results using NSE4A antibodies across different model systems, researchers should consider these methodological approaches:

• Epitope conservation analysis: Compare the antibody's target sequence (29-57 amino acids in the N-terminal region for some antibodies) across species to determine if epitope variations might affect antibody recognition . Sequence alignments of NSE4A orthologs can reveal critical differences that might affect antibody binding.

• Species-specific validation: Perform rigorous validation studies in each model organism using the techniques described in section 2.2, as antibody performance can vary substantially between species.

• Alternative antibody approaches: When possible, use multiple antibodies targeting different epitopes of NSE4A. For example, compare results between N-terminal and C-terminal targeting antibodies.

• Genetic complementation studies: In cases of discrepancy, use genetic approaches like expressing tagged versions of NSE4A (such as NSE4A::GFP fusion proteins) to confirm antibody observations .

• Context-dependent expression analysis: NSE4A expression and localization patterns may genuinely differ between tissues or developmental stages. In Arabidopsis, NSE4A expression has been detected across different tissue types, while NSE4B expression was limited primarily to generative tissue .

By systematically addressing these factors, researchers can resolve apparent contradictions and develop a more cohesive understanding of NSE4A biology across different experimental systems.

How can NSE4A antibodies contribute to research on DNA repair mechanisms?

NSE4A antibodies provide crucial tools for investigating DNA repair mechanisms, particularly homologous recombination pathways, through several methodological approaches:

• DNA damage response visualization: By combining NSE4A immunolabeling with markers of DNA double-strand breaks (such as γ-H2AX), researchers can track the recruitment of the SMC5/6 complex to sites of DNA damage. Studies have shown differential responses of NSE4A after induced DNA double-strand breaks, suggesting its involvement in repair processes .

• Co-immunoprecipitation (Co-IP) studies: NSE4A antibodies can be used to identify protein-protein interactions within the DNA repair machinery. This approach can reveal how NSE4A within the SMC5/6 complex interacts with other repair factors, such as the SMC1-SMC3 cohesin complex which it may recruit to double-strand breaks .

• Temporal dynamics analysis: Immunofluorescence studies using NSE4A antibodies can track the temporal recruitment and dissociation of repair factors during the DNA damage response, providing insights into the sequential assembly of repair complexes.

• Functional recovery assays: NSE4A antibodies can be used to monitor the restoration of normal chromatin organization after DNA damage, particularly in cellular models with defects in telomere maintenance like ALT cell lines .

These approaches collectively enable detailed investigation of NSE4A's role in maintaining genome integrity through DNA repair pathways.

What are the optimal protocols for using NSE4A antibodies in meiosis research?

For effective application of NSE4A antibodies in meiosis research, investigators should follow these specialized protocols:

• Sample preparation: For plant meiotic studies, follow established protocols for preparing pollen mother cells (PMCs) such as the method described by Armstrong and Osman (2013) . For animal models, adapt standard meiotic chromosome spreading techniques.

• Co-immunolabeling approach: For comprehensive analysis of meiotic processes, combine NSE4A antibodies (typically used at 1:250 dilution) with markers of meiotic structures such as the synaptonemal complex protein ZYP1 (typically used at 1:1000 dilution) .

• Sequential imaging strategy: To capture the dynamic localization of NSE4A throughout meiosis, systematically image cells at different meiotic stages from leptotene through tetrad formation. Research has shown that NSE4A signals are detectable in G2, leptotene, zygotene, and pachytene cells, may become weak or invisible in condensed metaphase I and anaphase I chromosomes, but recover in prophase II and tetrad stages .

• Cross-species comparison: When studying meiotic functions of NSE4A across different species, researchers should note that the Arabidopsis NSE4A antibody has been successfully used with Brassica rapa meiocytes, showing colocalization with the synaptonemal complex protein ZYP1 during pachytene .

• Validation with fluorescent protein fusions: Complement antibody-based studies with observations of fluorescently tagged NSE4A (such as NSE4A::GFP) to confirm localization patterns during meiosis. In transgenic Arabidopsis expressing pnse4A::gNse4A::GFP, NSE4A creates 'line-like' structures during prophase I that resemble ZYP1 patterns .

These methodological considerations ensure robust and reproducible investigation of NSE4A's meiotic functions across different model systems.

What are common challenges when using NSE4A antibodies and how can they be addressed?

Researchers working with NSE4A antibodies may encounter several technical challenges that can be systematically addressed:

• High background signal: This common issue may be resolved by:

  • Increasing blocking stringency (using 5% BSA instead of 3%)

  • Optimizing antibody dilution (starting with 1:1000 for Western blots and adjusting as needed)

  • Performing additional washing steps with PBS-T

  • Using antigen competition controls to distinguish specific from non-specific binding

• Weak or absent signals: When signal strength is insufficient:

  • Optimize protein extraction protocols specifically for nuclear proteins (where NSE4A is primarily located)

  • Reduce antibody dilution incrementally (e.g., from 1:1000 to 1:500)

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

  • Use signal amplification systems compatible with your detection method

• Inconsistent results across experiments: To improve reproducibility:

  • Standardize sample preparation protocols, particularly for nuclear extraction

  • Aliquot antibodies to avoid freeze-thaw cycles (following storage recommendations to maintain at 2-8°C for up to 2 weeks or at -20°C in small aliquots for long-term storage)

  • Include positive controls in each experiment

  • Maintain consistent blocking, washing, and incubation conditions

• Cross-reactivity issues: When specificity is a concern:

  • Verify the antibody's species reactivity before use (some NSE4A antibodies are human-specific)

  • Perform validation experiments as outlined in section 2.2

  • Consider using more specific monoclonal antibodies if available

By systematically addressing these challenges, researchers can optimize their experimental protocols for consistent and reliable results with NSE4A antibodies.

How are NSE4A antibodies being used to advance understanding of telomere biology?

NSE4A antibodies are becoming instrumental in exploring the frontier of telomere biology research through several innovative approaches:

• Alternative lengthening of telomeres (ALT) research: NSE4A antibodies help investigate how the SMC5/6 complex maintains telomeres via recombination in ALT cell lines. The complex has been shown to mediate sumoylation of shelterin complex components, which may lead to shelterin complex disassembly in ALT-associated PML bodies (APBs) .

• Telomeric chromatin organization studies: Immunolabeling with NSE4A antibodies, particularly using super-resolution microscopy techniques, enables detailed examination of protein localization at telomeres. Studies have confirmed NSE4A localization to chromosomal telomeres .

• Telomere damage response analysis: By combining NSE4A immunolabeling with markers of telomere dysfunction, researchers can investigate how the SMC5/6 complex responds to telomere-specific DNA damage.

• Comparative telomere biology: NSE4A antibodies enable cross-species comparison of telomere maintenance mechanisms. Studies in both plant and animal systems have revealed conservation of NSE4A's role in genome stability, though with species-specific adaptations .

These approaches collectively contribute to our understanding of how NSE4A and the SMC5/6 complex participate in maintaining telomere integrity, with implications for aging research and cancer biology.

What methodological considerations are important when using NSE4A antibodies to study protein-protein interactions within the SMC5/6 complex?

Investigating protein-protein interactions within the SMC5/6 complex using NSE4A antibodies requires sophisticated methodological approaches:

• Co-immunoprecipitation (Co-IP) optimization: When using NSE4A antibodies for Co-IP:

  • Use gentle lysis conditions to preserve native protein complexes

  • Consider crosslinking approaches to capture transient interactions

  • Include appropriate controls (IgG control, input samples)

  • Validate interactions through reciprocal Co-IP with antibodies against other complex components (SMC5, SMC6, NSE3)

• Proximity ligation assays (PLA): This technique can visualize protein-protein interactions in situ:

  • Combine NSE4A antibodies with antibodies against potential interaction partners

  • Optimize antibody concentrations to minimize background

  • Include appropriate negative controls (proteins known not to interact with NSE4A)

  • Quantify PLA signals to assess interaction strength

• Functional domain mapping: To understand the structural basis of interactions:

  • Use NSE4A antibodies in conjunction with domain-specific mutants

  • Compare interaction patterns between wild-type NSE4A and truncated or point-mutated variants

  • Consider combining with structural prediction approaches

• Dynamic interaction analysis: To study interaction kinetics:

  • Use NSE4A antibodies to track complex assembly/disassembly during cell cycle progression

  • Compare interaction patterns before and after DNA damage induction

  • Consider live-cell imaging approaches using fluorescently tagged components alongside validation with antibodies

• Cross-species conservation analysis: When studying evolutionary conservation of interactions:

  • Test whether NSE4A antibodies recognize orthologs in different species

  • Compare interaction networks across evolutionary diverse models

  • Align with sequence conservation data to identify functionally critical interaction interfaces

These methodological considerations enable comprehensive analysis of NSE4A's role within the SMC5/6 complex architecture and its functional interactions during DNA repair and chromatin organization processes.