SEZ6 Antibody, Biotin conjugated

What is SEZ6 and why is it significant in neurological research?

SEZ6 (Seizure Related 6 Homolog) is a transmembrane protein discovered on the surface of selected neuronal lineage cells. Its significance in neurological research stems from its role as a novel trafficking protein for kainate receptors (KARs), controlling their activity, localization, and glycosylation. The extracellular domain of SEZ6 can bind GluK2 and controls the complex N-glycosylation of GluK2 and/or GluK3 that occurs late in the secretory pathway. This function appears to be mediated predominantly by the full-length form of SEZ6, rather than its cleaved form, and is independent of BACE1 cleavage. This unique regulatory role establishes SEZ6 as a critical component in the regulation of KAR activity .

What are the specific characteristics of biotin-conjugated SEZ6 antibodies?

Biotin-conjugated SEZ6 antibodies are polyclonal antibodies typically derived from rabbit hosts that specifically target human SEZ6. These antibodies specifically recognize amino acids 20-267 of the SEZ6 protein. The biotin conjugation enables enhanced detection in various immunoassays through the strong biotin-streptavidin interaction. Commercial preparations are typically purified using Protein G chromatography with a purity exceeding 95%. The antibodies are supplied in liquid format with a buffer containing 0.03% Proclin 300 as a preservative, 50% glycerol, and 0.01M PBS at pH 7.4 .

What are the optimal conditions for using biotin-conjugated SEZ6 antibodies in ELISA?

When using biotin-conjugated SEZ6 antibodies in ELISA applications, researchers should consider the following protocol optimizations:

• Dilution optimization: The working dilution should be empirically determined for each specific experimental setup, typically starting with a range of 1:500 to 1:2000.

• Detection system: Utilize streptavidin-HRP conjugates with appropriate detection substrates such as TMB (3,3',5,5'-Tetramethylbenzidine).

• Blocking conditions: Use 3-5% BSA in PBS or 5% non-fat dry milk to reduce background.

• Incubation parameters: Optimal results are typically achieved with primary antibody incubation at 4°C overnight or at room temperature for 1-2 hours.

• Washing stringency: Perform 3-5 washes with PBS-T (0.05% Tween-20) between steps to minimize non-specific binding.

The specific affinity for the SEZ6 epitope (AA 20-267) makes these antibodies particularly suitable for detecting both native and recombinant human SEZ6 proteins. Researchers should validate the antibody performance with positive and negative controls relevant to their experimental system .

How can SEZ6 antibodies be utilized to investigate neuronal plasma membrane trafficking?

To investigate neuronal plasma membrane trafficking using SEZ6 antibodies, researchers can implement a multi-faceted approach:

• Surface biotinylation assays: Use cell-impermeable biotinylation reagents to label surface proteins, followed by precipitation with streptavidin beads and immunoblotting with SEZ6 antibodies to quantify surface expression levels.

• SUSPECS (Surface protein biotinylation and systematic mass spectrometry) analysis: This technique effectively enriches for membrane proteins, with 40% of all detected proteins and 90% of glycosylated proteins classified as membrane proteins according to UniProt keywords. This approach has been validated in SEZ6 knockout (SEZ6KO) versus wild-type neuron comparisons .

• Immunocytochemistry with and without permeabilization: Compare staining patterns to differentiate between surface and intracellular pools of SEZ6 and its binding partners.

• Live-cell imaging: Use fluorescently labeled SEZ6 antibodies to track real-time changes in surface expression and trafficking.

• Co-immunoprecipitation studies: Employ SEZ6 antibodies to investigate interactions with GluK2/GluK3 and other trafficking components.

These approaches can reveal how SEZ6 regulates the transport of KAR subunits GluK2 and/or GluK3 to the neuronal plasma membrane, offering insights into synaptic transmission mechanisms .

What controls should be included when validating SEZ6 antibody specificity?

When validating the specificity of SEZ6 antibodies, the following controls should be implemented:

• Genetic controls: Include samples from SEZ6 knockout (SEZ6KO) neurons as negative controls. Consistent surface detection in wild-type neurons but not in SEZ6KO neurons confirms antibody specificity .

• Peptide competition assays: Pre-incubate the antibody with excess target peptide (AA 20-267) to block specific binding sites and demonstrate binding specificity.

• Cross-reactivity assessment: Test against other SEZ6 family members (SEZ6L and SEZ6L2, which share >40% identity and 60% similarity with SEZ6) to ensure the antibody does not cross-react with these related proteins .

• Isoform detection profile: Evaluate the antibody's ability to detect different SEZ6 isoforms (types I, II, and III) resulting from mRNA alternative splicing. Types I and II are cell surface proteins with a single transmembrane domain, while type III is a secreted isoform (sSez6) .

• Western blot analysis: Verify the detection of proteins at the expected molecular weight, considering post-translational modifications such as glycosylation that may affect migration patterns.

These validation steps ensure experimental results can be confidently attributed to specific detection of SEZ6, minimizing the risk of misinterpreting data due to non-specific binding or cross-reactivity.

How can biotin-conjugated SEZ6 antibodies be employed in cancer biomarker studies?

Biotin-conjugated SEZ6 antibodies offer significant utility in cancer biomarker studies, particularly for small cell lung cancer (SCLC) research, through several methodological approaches:

The high specificity of these antibodies for human SEZ6 makes them valuable tools for translational cancer research, particularly in developing targeted therapeutic approaches for SCLC .

What methodology can be used to investigate SEZ6's role in receptor glycosylation?

To investigate SEZ6's role in receptor glycosylation, particularly of GluK2/GluK3 kainate receptors, researchers can implement the following methodological approach:

• Glycosidase treatment assays: Compare the electrophoretic mobility of GluK2/GluK3 from wild-type and SEZ6-deficient samples before and after treatment with specific glycosidases (PNGase F, Endo H) to assess differences in N-glycosylation patterns.

• Lectin binding studies: Use different plant lectins with specificity for particular glycan structures to profile the glycosylation states of GluK2/GluK3 in the presence or absence of SEZ6.

• Pulse-chase experiments: Track the maturation of newly synthesized GluK2/GluK3 in SEZ6-expressing and SEZ6-knockout cells to determine how SEZ6 affects the rate and extent of complex glycosylation.

• Site-directed mutagenesis: Modify potential N-glycosylation sites in GluK2/GluK3 to identify which specific sites are influenced by SEZ6 activity.

• Co-immunoprecipitation with glycosylation enzymes: Investigate whether SEZ6 interacts with specific glycosyltransferases in the Golgi apparatus to facilitate GluK2/GluK3 glycosylation.

This approach can reveal how SEZ6 specifically controls complex N-glycosylation of GluK2/GluK3 that occurs late in the secretory pathway, a process critical for proper receptor trafficking and function at the neuronal plasma membrane .

How do BACE1 inhibitors affect SEZ6 function in neuronal trafficking studies?

When investigating how BACE1 (Beta-site APP Cleaving Enzyme 1) inhibitors affect SEZ6 function in neuronal trafficking studies, researchers should consider the following methodological approach:

• Proteolytic processing analysis: Compare the ratio of full-length SEZ6 to its cleaved fragments in the presence and absence of BACE1 inhibitors using western blotting with domain-specific antibodies.

• Surface expression quantification: Monitor changes in surface expression of SEZ6 and its cargo proteins (particularly GluK2/GluK3) following BACE1 inhibitor treatment using surface biotinylation or immunofluorescence techniques.

• Trafficking kinetics assessment: Use pulse-chase experiments with surface labeling to determine if BACE1 inhibition alters the rate of SEZ6-dependent trafficking of kainate receptors to the plasma membrane.

• Functional electrophysiology: Record kainate receptor-mediated currents in neurons treated with BACE1 inhibitors to evaluate functional consequences of altered trafficking.

• Domain-specific mutant studies: Compare trafficking functions of wild-type SEZ6 versus cleavage-resistant SEZ6 mutants to delineate which functions require proteolytic processing.

Current research indicates that SEZ6's role in controlling GluK2/GluK3 complex N-glycosylation and trafficking appears to be independent of BACE1 cleavage, being mediated predominantly by the full-length form of SEZ6 rather than its cleaved form. This suggests that BACE1 inhibitors, while preventing SEZ6 cleavage, might not directly impair its trafficking functions regarding kainate receptors .

What are the optimal storage conditions for maintaining biotin-conjugated SEZ6 antibody activity?

To maintain optimal activity of biotin-conjugated SEZ6 antibodies, the following storage conditions should be implemented:

• Temperature requirements: Store at -20°C or -80°C for long-term stability. The choice between these temperatures depends on anticipated frequency of use, with -80°C being preferred for extended storage periods .

• Aliquoting strategy: Upon receipt, divide the antibody into small single-use aliquots to minimize freeze-thaw cycles. Each freeze-thaw event can reduce antibody activity by approximately 10-15% .

• Freeze-thaw management: Avoid repeated freezing and thawing of the same aliquot. The manufacturer's recommendation specifically warns against this practice .

• Buffer composition: The commercial formulation (50% glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as preservative) is designed to maintain stability during storage. Do not dilute the stock solution prior to storage as this can compromise stability .

• Handling precautions: Note that the preservative (Proclin 300) is classified as a hazardous substance and should be handled by trained personnel using appropriate safety measures .

Following these guidelines will help ensure consistent antibody performance throughout your research project, minimizing variability due to reagent degradation.

How can researchers optimize signal-to-noise ratio when using biotin-conjugated SEZ6 antibodies?

To optimize the signal-to-noise ratio when using biotin-conjugated SEZ6 antibodies, researchers should implement the following technical strategies:

• Titration optimization: Systematically test a range of antibody dilutions (typically starting from 1:100 to 1:5000) to identify the concentration that provides maximum specific signal with minimal background. The optimal working dilution should be determined empirically for each specific application and experimental system .

• Blocking protocol refinement: Use freshly prepared blocking solutions (3-5% BSA or 5% non-fat milk) and extend blocking time to 1-2 hours at room temperature to reduce non-specific binding sites before antibody application.

• Endogenous biotin blocking: For tissues or cells with high endogenous biotin (e.g., liver, kidney), implement an avidin/biotin blocking step before antibody incubation to prevent false-positive signals.

• Detection system selection: Choose streptavidin conjugates with appropriate sensitivity for your application; HRP-based systems can be enhanced with tyramide signal amplification for detecting low-abundance targets, while fluorophore-conjugated streptavidin offers better spatial resolution for microscopy.

• Wash optimization: Increase washing stringency (more cycles, longer duration, or higher detergent concentration) to reduce background, especially in high-sensitivity applications.

• Control inclusion: Always run parallel negative controls (omitting primary antibody, using non-immunized IgG, testing on SEZ6-negative samples) to accurately assess background levels.

These methodological refinements help ensure that signals detected truly represent SEZ6 expression rather than technical artifacts, critical for both qualitative and quantitative analyses.

What are the common pitfalls when working with SEZ6 antibodies and how can they be mitigated?

When working with SEZ6 antibodies, researchers should be aware of these common pitfalls and their mitigation strategies:

• Cross-reactivity with SEZ6 family members: SEZ6 shares >40% identity and 60% similarity with other family members (SEZ6L and SEZ6L2) . To mitigate this:

  • Validate antibody specificity using SEZ6 knockout models or siRNA knockdown systems

  • Perform peptide competition assays with specific SEZ6 peptides

  • Consider using multiple antibodies targeting different epitopes of SEZ6

• Isoform detection variability: SEZ6 exists in multiple isoforms due to alternative splicing (two transmembrane forms and one secreted form) . To address this:

  • Confirm which isoforms your antibody detects through western blotting

  • Select antibodies with epitopes present in your isoform of interest

  • Use PCR to verify isoform expression in your experimental system

• Glycosylation interference: SEZ6 is heavily glycosylated, which can mask epitopes and affect antibody binding. To overcome this:

  • Test antibody recognition of both native and deglycosylated samples

  • Consider epitope exposure techniques if targeting regions affected by glycosylation

• Fixation sensitivity: Some epitopes may be sensitive to certain fixation protocols. To manage this:

  • Compare antibody performance across different fixation methods

  • Test both immersion and perfusion fixation for tissue samples

  • Consider antigen retrieval methods to restore epitope accessibility

• Low abundance detection challenges: SEZ6 may be expressed at low levels in some tissues . For improved detection:

  • Implement signal amplification systems (e.g., tyramide signal amplification)

  • Increase antibody incubation time to enhance binding

  • Concentrate protein samples when possible for biochemical applications

By anticipating these challenges and implementing appropriate controls and optimizations, researchers can improve the reliability and reproducibility of experiments using SEZ6 antibodies.

How might SEZ6 antibodies contribute to developing targeted therapeutics for small cell lung cancer?

SEZ6 antibodies are poised to make significant contributions to targeted therapeutics for small cell lung cancer (SCLC) through several methodological approaches:

• ADC development validation: Biotin-conjugated SEZ6 antibodies can be used to validate the expression, accessibility, and internalization kinetics of SEZ6 in patient-derived SCLC samples, providing crucial data for antibody-drug conjugate (ADC) development. Recent clinical trials of ADCs targeting SEZ6 have shown promising results, with AbbVie's ABBV-706 demonstrating a 60.9% confirmed objective response rate in SCLC patients .

• Patient stratification methodology: Developing immunohistochemistry protocols using SEZ6 antibodies can help stratify SCLC patients based on SEZ6 expression levels, potentially identifying those most likely to respond to SEZ6-targeted therapies.

• Resistance mechanism investigation: As SEZ6-targeted therapies advance clinically, SEZ6 antibodies will be essential tools for studying acquired resistance mechanisms, such as receptor downregulation, mutation, or compensatory pathway activation.

• Combination therapy rational design: By using SEZ6 antibodies to monitor how receptor expression and trafficking change in response to conventional chemotherapy or immunotherapy, researchers can develop rational combination approaches that maximize therapeutic efficacy.

• Circulating tumor cell monitoring: SEZ6 antibodies can facilitate the development of liquid biopsy approaches to monitor treatment response and disease progression in real-time through detection of SEZ6-positive circulating tumor cells.

The differential expression pattern of SEZ6—limited in normal tissues but highly expressed in SCLC—makes it an ideal target for selective cancer therapy, potentially offering improved therapeutic windows compared to other targets .

What methodologies can evaluate the role of SEZ6 in neurodevelopmental disorders?

To evaluate the role of SEZ6 in neurodevelopmental disorders, researchers can implement these methodological approaches:

• Temporal expression profiling: Using SEZ6 antibodies for immunohistochemistry and western blotting across developmental timepoints to map normal expression patterns and identify aberrations in disorder models. This approach can reveal critical developmental windows where SEZ6 dysfunction may contribute to pathology.

• High-resolution subcellular localization: Employing super-resolution microscopy with SEZ6 antibodies co-labeled with synaptic markers to assess SEZ6's precise distribution at developing synapses and how this may be altered in disorder models.

• Functional circuit analysis: Combining SEZ6 immunolabeling with electrophysiological recordings to correlate receptor trafficking defects with functional synaptic transmission abnormalities in specific neural circuits implicated in neurodevelopmental disorders.

• Kainate receptor glycosylation assessment: Developing mass spectrometry-based glycoproteomics workflows to characterize how SEZ6 dysfunction affects the complex glycosylation pattern of GluK2/GluK3 kainate receptors during critical developmental periods.

• Behavioral phenotyping with molecular correlation: Performing systematic behavioral assessment of SEZ6 knockout or mutant models, followed by region-specific molecular analysis using SEZ6 antibodies to establish direct links between molecular dysfunction and behavioral abnormalities.

• Conditional and cell-type specific manipulation: Utilizing Cre-lox systems for temporal and cell-type specific SEZ6 knockout, followed by immunohistochemical analysis to dissect the cell-autonomous versus non-cell-autonomous effects of SEZ6 dysfunction.

Given SEZ6's critical role in controlling the activity, localization, and glycosylation of kainate receptors—which are implicated in several neurodevelopmental disorders—these methodological approaches can yield significant insights into pathological mechanisms and potential therapeutic strategies .

How can researchers investigate the interplay between SEZ6 and other neuronal trafficking proteins?

To investigate the interplay between SEZ6 and other neuronal trafficking proteins, researchers should implement the following comprehensive methodological approach:

• Proximity-based proteomics: Use BioID or APEX2 proximity labeling techniques, where biotin ligase is fused to SEZ6, to identify proteins that exist in close proximity to SEZ6 within the trafficking machinery. This approach can reveal novel interaction partners beyond the known GluK2/GluK3 interactions .

• Co-immunoprecipitation network analysis: Perform sequential co-immunoprecipitation experiments using biotin-conjugated SEZ6 antibodies followed by mass spectrometry to map the protein interaction network. Compare these networks between wild-type and neurological disorder models to identify dysregulated interactions.

• Live trafficking dynamics: Implement dual-color live-cell imaging using differentially labeled SEZ6 and candidate trafficking proteins to analyze their temporal and spatial co-localization during vesicular transport events in neuronal processes.

• Domain-specific interaction mapping: Create a series of SEZ6 domain deletion constructs to determine which structural features (e.g., which of the 5 sushi domains or 2-3 CUB domains) are responsible for interactions with specific trafficking machinery components .

• Competitive binding assays: Use purified components and surface plasmon resonance or microscale thermophoresis to quantitatively assess binding affinities between SEZ6 and various trafficking proteins, and determine if they compete for the same binding sites.

• Conditional knockout compensatory analysis: Analyze changes in expression and localization of other trafficking proteins when SEZ6 is conditionally deleted, to identify compensatory mechanisms and functional redundancies within the trafficking machinery.

This multipronged approach can elucidate how SEZ6 functions within the broader context of neuronal protein trafficking, potentially revealing novel therapeutic targets for neurological disorders associated with protein trafficking defects.