NPSN13 Antibody

What is NPSN13 and what is its role in plant cells?

NPSN13 (Novel Plant SNARE 13) is a member of the plant-specific SNARE protein family involved in membrane fusion processes. It functions as a paralog to NPSN11 and NPSN12, with roles primarily in membrane trafficking during plant cell division. While the specific function of NPSN13 remains under investigation, research indicates it likely participates in SNARE complexes similar to NPSN11, which has been shown to interact with other SNAREs such as KNOLLE and SYP71 during cytokinesis . These complexes are essential for proper formation of the cell plate during plant cell division, progressing from the center to the periphery of dividing cells. Understanding NPSN13's function requires consideration of the complete SNARE complex network in plants, where multiple complexes with different compositions may contribute to the same cellular processes.

What are the key characteristics of a high-quality NPSN13 antibody?

A high-quality NPSN13 antibody should demonstrate several critical properties: (1) High specificity, with minimal cross-reactivity to related plant SNARE proteins like NPSN11 and NPSN12; (2) Appropriate sensitivity to detect endogenous levels of NPSN13 in plant samples; (3) Consistent performance across multiple experimental techniques including Western blotting, immunoprecipitation, and immunolocalization; and (4) Validation in the specific plant species being studied. The antibody should be tested for its ability to recognize both native and denatured forms of NPSN13, depending on the intended application. Researchers should verify antibody performance through positive and negative controls, including genetic knockouts of NPSN13 where available, to ensure reliable detection of the target protein.

How can I distinguish between NPSN13, NPSN11, and NPSN12 in experimental settings?

Distinguishing between these closely related SNARE proteins requires careful experimental design. First, utilize antibodies raised against unique epitopes specific to each NPSN protein. Synthetic peptides corresponding to non-conserved regions of these proteins can serve as immunogens to produce highly specific antibodies . Second, implement comparative Western blot analysis with recombinant proteins as standards to confirm molecular weight differences. Third, perform immunoprecipitation experiments with tagged versions of each protein to verify specific antibody recognition. Fourth, conduct immunofluorescence microscopy to determine potential differences in subcellular localization patterns. Finally, validate results using genetic approaches with knockout or knockdown lines for each NPSN protein to confirm antibody specificity. When analyzing results, pay particular attention to potential shifts in band patterns that might indicate cross-reactivity among these paralogous proteins.

What are the optimal conditions for using NPSN13 antibodies in Western blotting of plant samples?

The optimal conditions for Western blotting with NPSN13 antibodies require careful sample preparation and protocol optimization. For plant tissue extraction, use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail. Mechanical disruption at 4°C followed by centrifugation at 14,000g for 15 minutes yields suitable protein extracts. For protein separation, 12-15% SDS-PAGE gels provide optimal resolution for NPSN13 (approximately 30 kDa). Transfer to PVDF membranes at 100V for 1 hour in a standard Towbin buffer system. Block with 5% non-fat dry milk in TBST for 1 hour at room temperature. For primary antibody incubation, use NPSN13 antibody at 1:1000 dilution overnight at 4°C, followed by HRP-conjugated secondary antibody at 1:5000 for 1 hour at room temperature. Enhanced chemiluminescence detection typically yields optimal results. Include positive controls (recombinant NPSN13) and negative controls (NPSN13 knockout samples) to validate antibody specificity and performance.

How can I optimize immunoprecipitation protocols when studying NPSN13 interactions with other SNARE proteins?

Optimizing immunoprecipitation (IP) protocols for NPSN13 interaction studies requires careful consideration of detergents, buffers, and experimental controls. Begin with a mild lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40 or 0.5% digitonin) to preserve native protein interactions. Pre-clear lysates with Protein A/G beads for 1 hour to reduce non-specific binding. For the IP reaction, use 2-5 μg of NPSN13 antibody per 500 μg of total protein and incubate overnight at 4°C with gentle rotation. Capture antibody-protein complexes with 30 μl of Protein A/G magnetic beads for 2 hours. Perform five washes with decreasing salt concentrations to remove non-specific interactions while preserving specific complexes. When analyzing potential SNARE partners, cross-reference with known interaction patterns from related proteins like NPSN11, which forms distinct complexes with KNOLLE but not with SNAP33 . Include reciprocal IPs and isotype control antibodies to validate interactions. For detecting weak or transient interactions, consider chemical crosslinking prior to cell lysis or proximity-based labeling approaches.

What are the key considerations for generating recombinant NPSN13 protein for antibody validation?

Generating high-quality recombinant NPSN13 protein requires careful consideration of expression systems, purification strategies, and quality control measures. First, design constructs that exclude the transmembrane domain to improve solubility while preserving key epitopes. Consider using bacterial expression systems (E. coli BL21 or Rosetta strains) with fusion tags (His6, GST, or MBP) to enhance solubility and facilitate purification. Expression at lower temperatures (16-18°C) after IPTG induction (0.1-0.5 mM) often improves proper folding. For purification, implement a two-step approach using affinity chromatography followed by size exclusion chromatography to ensure high purity. Verify protein identity through mass spectrometry and N-terminal sequencing. Assess protein quality through circular dichroism to confirm proper secondary structure. When using the recombinant protein for antibody validation, create a dilution series (1-100 ng) for Western blotting to establish detection limits. Additionally, pre-absorb antibodies with the recombinant protein to confirm specificity in immunolocalization experiments, noting that antibody performance may differ between denatured and native conformations of NPSN13.

How should I interpret conflicting immunolocalization results with NPSN13 antibodies?

Conflicting immunolocalization results with NPSN13 antibodies can stem from multiple methodological and biological factors that require systematic troubleshooting. First, evaluate fixation methods as different protocols can significantly affect epitope accessibility—compare paraformaldehyde (4%) with glutaraldehyde (0.1-0.5%) fixation. Second, test various antigen retrieval techniques including heat-induced epitope retrieval and enzymatic treatments. Third, assess antibody specificity using absorption controls with recombinant NPSN13 protein and validate with genetic knockouts or knockdowns. Fourth, compare polyclonal versus monoclonal antibodies, as they recognize different epitopes. Fifth, consider developmental and tissue-specific expression patterns, as NPSN13 localization may change during cell division or differentiation. Based on SNARE protein dynamics observed with related proteins, NPSN13 likely shuttles between different membrane compartments and the cell plate during cytokinesis . Cross-reference findings with fluorescently tagged NPSN13 in transgenic plants to resolve discrepancies. Document all experimental conditions meticulously, as seemingly minor variations in protocols can profoundly affect results.

What control experiments are essential when publishing research using NPSN13 antibodies?

Publishing rigorous research with NPSN13 antibodies requires comprehensive controls to ensure data reliability and reproducibility. Essential controls include: (1) Antibody specificity validation using Western blots comparing wild-type and NPSN13 knockout/knockdown plant samples; (2) Peptide competition assays demonstrating signal reduction when antibodies are pre-incubated with immunizing peptides; (3) Cross-reactivity assessment against recombinant NPSN11 and NPSN12 proteins; (4) Secondary antibody-only controls to identify non-specific binding; (5) Positive controls using tagged NPSN13 constructs detected with both tag-specific and NPSN13-specific antibodies; (6) Biological replicates across different developmental stages and tissue types to account for expression variability; (7) Technical replicates using different antibody lots to ensure consistency; and (8) Complementary approaches such as RNA expression analysis or fluorescent protein fusions to corroborate antibody-based findings. For co-localization studies, include appropriate markers for cellular compartments and quantitative colocalization metrics. These comprehensive controls establish a foundation for reliable interpretation of results and facilitate acceptance by the scientific community.

How can I resolve inconsistencies between Western blot and immunohistochemistry results when using NPSN13 antibodies?

Inconsistencies between Western blot and immunohistochemistry (IHC) results often reflect fundamental differences in how antibodies interact with proteins in these distinct methodologies. To resolve such discrepancies: First, evaluate epitope accessibility—Western blots detect denatured proteins while IHC typically preserves partial native structure. Try different fixation protocols (acetone, methanol, or paraformaldehyde) and antigen retrieval methods for IHC. Second, assess antibody concentration optimization independently for each technique; Western blots typically require higher dilutions (1:1000-1:5000) than IHC (1:50-1:500). Third, validate antibody specificity in both applications using knockout controls and peptide competition assays. Fourth, consider post-translational modifications that might differ between sample preparations. Fifth, evaluate protein-protein interactions that may mask epitopes in IHC but not in denaturing Western blots. Based on data from other SNARE proteins, NPSN13 likely forms complexes with partners that could affect antibody binding . Document protein extraction methods carefully, as membrane protein solubilization requires specific detergents that may differentially impact epitope preservation. Complementary techniques such as proximity ligation assays can help bridge observations from these different methodologies.

How can I implement proximity labeling techniques with NPSN13 antibodies to identify novel interaction partners?

Implementing proximity labeling with NPSN13 antibodies requires strategic experimental design to capture the dynamic SNARE interactome. Begin by generating recombinant constructs expressing NPSN13 fused to proximity labeling enzymes such as BioID2 (biotin ligase) or APEX2 (ascorbate peroxidase) under native promoters using CRISPR-Cas9 genome editing in Arabidopsis. For antibody-based approaches, conjugate NPSN13 antibodies directly to APEX2 using commercial conjugation kits with optimized molar ratios. Perform labeling reactions in planta by providing biotin-phenol substrate and triggering labeling with hydrogen peroxide for 1 minute—this short reaction time captures transient interactions common in SNARE complex assembly. Extract biotinylated proteins using stringent lysis conditions (1% SDS followed by dilution to 0.1%) and capture with streptavidin beads. Analyze the interactome using quantitative mass spectrometry, comparing results to controls including non-transfected plants and plants expressing only the labeling enzyme. Cross-reference findings with known SNARE interaction networks, particularly focusing on proteins that interact with NPSN11 and NPSN12 . Validate top candidates through reciprocal immunoprecipitation and functional studies using conditional knockout approaches. This method can reveal both stable and transient interactions within the NPSN13 interaction network.

What are the most effective approaches for studying NPSN13 dynamics during plant cell division using super-resolution microscopy?

Studying NPSN13 dynamics during plant cell division requires integration of advanced microscopy techniques with careful sample preparation and image analysis. For super-resolution imaging, implement Structured Illumination Microscopy (SIM) or Stimulated Emission Depletion (STED) microscopy, which can achieve 50-100 nm resolution to resolve individual vesicles and membrane subdomains. Prepare samples using either (1) immunolocalization with validated NPSN13 antibodies in fixed cells or (2) fluorescent protein fusions for live-cell imaging. For fixed-cell approaches, use 4% paraformaldehyde with 0.1% glutaraldehyde for 20 minutes, followed by enzymatic cell wall digestion to improve antibody penetration. For live-cell imaging, generate stable Arabidopsis lines expressing NPSN13-mNeonGreen under native promoters using CRISPR-Cas9 knock-in strategies. Implement multi-color imaging to simultaneously visualize NPSN13 with other SNARE partners and markers for specific membrane compartments. For temporal resolution, acquire images every 15-30 seconds during active cytokinesis. Apply deconvolution algorithms optimized for plant cells to improve image quality. Quantify protein dynamics using fluorescence recovery after photobleaching (FRAP) or photoactivatable fluorescent proteins to measure protein turnover rates at the cell plate. Compare NPSN13 dynamics with those of NPSN11, which has been shown to participate in tetrameric SNARE complexes with SYP71 and VAMP721/722 during cytokinesis .

How can quantitative proteomics be integrated with NPSN13 antibody-based purification to map SNARE complex stoichiometry?

Integrating quantitative proteomics with NPSN13 antibody-based purification enables precise mapping of SNARE complex stoichiometry and dynamics. Begin with optimized immunoprecipitation using covalently coupled NPSN13 antibodies to high-capacity beads (NHS-activated magnetic beads) to minimize antibody contamination in downstream analysis. Implement SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling to compare protein abundances across different conditions, such as developmental stages or stress responses. For membrane protein extraction, use a combination of detergents (0.5% digitonin followed by 1% DDM) to preserve native complexes while ensuring efficient solubilization. Process samples using Filter-Aided Sample Preparation (FASP) with sequential digestion using LysC followed by trypsin to maximize peptide coverage. Analyze samples using high-resolution mass spectrometry with long chromatographic gradients (>120 minutes) to enhance separation of complex peptide mixtures. For data analysis, implement targeted quantification of SNARE proteins using parallel reaction monitoring (PRM) to achieve attomole sensitivity. Cross-reference stoichiometric data with structural models of SNARE complexes based on similar proteins like KNOLLE-containing complexes . Integrate this information with interaction dynamics data from crosslinking mass spectrometry (XL-MS) to generate comprehensive models of NPSN13-containing SNARE complex assembly, stability, and disassembly during membrane fusion events.

How do antibody epitope selections differ when targeting NPSN13 versus NPSN11 or NPSN12?

Effective epitope selection for differentiating between NPSN family members requires comprehensive sequence analysis and strategic immunogen design. These paralogous proteins share approximately 65-75% sequence identity in their functional domains, necessitating careful targeting of unique regions. For NPSN13-specific antibodies, the optimal approach targets the N-terminal variable region (amino acids 25-45) and the linker region between the SNARE domain and transmembrane domain (amino acids 210-230), which show the highest sequence divergence. In contrast, NPSN11 antibodies should target its unique C-terminal extension, which is absent in NPSN12 and NPSN13. For NPSN12, the central region of its SNARE domain contains distinctive residues suitable for specific antibody generation. When designing immunizing peptides, incorporate unique post-translational modification sites where possible, as these can differ substantially between paralogs. Validate epitope accessibility using protein structure prediction tools and consider surface exposure in the native protein conformation. Test candidate antibodies against recombinant versions of all three proteins to quantify cross-reactivity. In published studies examining NPSN11, researchers have successfully used antibodies targeting unique regions that do not cross-react with other family members, demonstrating the feasibility of this approach .

What can we learn about NPSN13 function from comparative studies with NPSN11 in SNARE complex formation?

Comparative studies of NPSN13 and NPSN11 SNARE complex formation provide critical insights into functional specialization and redundancy. Research has shown that NPSN11 participates in a tetrameric SNARE complex with Qc-SNARE SYP71 and R-SNARE VAMP721/722 during cytokinesis, while not interacting with SNAP33 . This suggests a model where NPSN13 might similarly form distinct complexes with specific Q- and R-SNAREs. Systematic co-immunoprecipitation experiments comparing NPSN13 and NPSN11 interactomes can reveal shared and unique binding partners. Unlike NPSN11, which is specifically involved in cytokinesis, NPSN13 may participate in additional membrane trafficking events during interphase or specialized responses. Analysis of mutant phenotypes is particularly informative—NPSN11 single mutants show mild cytokinesis defects, but double mutants with SYP71 exhibit severe cell division abnormalities . This suggests functional redundancy within SNARE complex networks, where NPSN13 might compensate for NPSN11 loss in certain contexts. Quantitative proteomics comparing wild-type, npsn11, and npsn13 mutants can reveal compensatory changes in SNARE protein expression and complex formation. Time-resolved studies of protein localization during cell division are essential, as NPSN11 shows specific recruitment to the cell plate, a pattern that may differ for NPSN13 depending on its functional specialization.

How does antibody affinity differ when detecting NPSN13 in different plant species, and what are the implications for cross-species research?

Antibody affinity for NPSN13 varies considerably across plant species due to evolutionary divergence in protein sequence and structure, creating significant implications for cross-species research. NPSN proteins emerged early in plant evolution but show variable conservation patterns—while the SNARE domain is generally well-preserved (80-90% identity within flowering plants), N- and C-terminal regions can diverge substantially (as low as 40-50% identity). This divergence affects antibody performance in several critical ways. First, antibodies raised against Arabidopsis NPSN13 typically show highest affinity for closely related Brassicaceae family members but demonstrate reduced sensitivity in monocots or non-vascular plants. Second, post-translational modifications differ significantly between species, affecting epitope accessibility and antibody recognition. Third, protein abundance varies across species, requiring optimization of antibody concentration and detection methods. To address these challenges, researchers should implement a multi-faceted approach: (1) Test antibodies against recombinant NPSN13 from multiple species to quantify affinity differences; (2) Identify conserved epitopes through comprehensive sequence alignment of NPSN13 orthologs; (3) Develop species-specific antibodies for critical model organisms; and (4) Validate antibody performance in each species using genetic controls. For evolutionary studies, targeting the most conserved regions of NPSN13 enables broader cross-species application, while species-specific antibodies provide higher sensitivity for detailed mechanistic investigations.