SQE6 Antibody

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

SEZ6 (Seizure protein 6) is a neuronal protein widely expressed throughout the brain that plays crucial roles in synaptic development and function, cell-cell recognition, and neuronal membrane signaling. The significance of SEZ6 in neuroscience stems from its essential role in maintaining the balance between dendrite elongation and branching during the development of complex dendritic arbors, as well as its involvement in establishing appropriate excitatory synaptic connectivity .

SEZ6 exists in three distinct splice isoforms: two transmembrane protein variants and one truncated secreted isoform. These isoforms demonstrate opposing functions in dendritic development, with the transmembrane form inhibiting outgrowth while the secreted form promotes it . Studies using knockout models have demonstrated that SEZ6 deficiency results in neurons with altered morphology (including increased dendrite numbers and reduced spine densities), modified electrophysiological properties, and deficits in memory, learning, and motor function .

Furthermore, SEZ6 has been implicated in various neurological and psychiatric conditions, with gene mutations and altered expression associated with febrile seizures, autism spectrum disorder, intellectual disability, developmental delay, and childhood-onset schizophrenia .

What types of SEZ6 antibodies are available for neuroscience research?

Several types of SEZ6 antibodies have been developed for research applications, including:

These antibodies are designed to target specific epitopes of the SEZ6 protein and have been validated for particular applications and species. The extracellular-targeting antibody (#ANR-206) recognizes an epitope corresponding to amino acid residues 849-863 of human SEZ6 (Accession Q53EL9) , allowing researchers to study the protein at the cell surface.

What are the optimal experimental conditions for SEZ6 antibody applications?

For Western blot applications, optimal experimental conditions include:

• Antibody dilution: 1:200 for Anti-SEZ6 (extracellular) Antibody and 1:1000 for Anti-Sez6 monoclonal antibody

• Sample preparation: Brain lysates from mouse or rat are commonly used

• Expected molecular weight: Approximately 170 kDa observed band size (107 kDa predicted)

• Blocking solution: 5% non-fat dry milk in TBST

• Secondary antibody: Species-appropriate HRP-conjugated antibody (e.g., Goat Anti-Rat IgG H&L for the monoclonal antibody)

For immunohistochemistry applications:

• Antibody dilution: 1:300 for Anti-SEZ6 (extracellular) Antibody

• Sample preparation: Perfusion-fixed frozen brain sections

• Detection system: Fluorescent secondary antibodies (e.g., goat anti-rabbit-AlexaFluor-488)

• Counterstaining: DAPI for nuclear visualization

• Positive control tissues: Substantia nigra pars compacta, temporal cortex, and piriform cortex

When conducting specificity controls, pre-incubation of the antibody with the corresponding blocking peptide (e.g., SEZ6 (extracellular) Blocking Peptide BLP-NR206) should suppress staining .

What biological samples are suitable for SEZ6 antibody analysis?

SEZ6 antibodies have been successfully employed with various biological samples:

• Brain tissue lysates from multiple species:

  • Mouse brain (whole and region-specific)

  • Rat brain (whole and region-specific)

  • Human neuroblastoma cell line (SH-SY5Y)

• Fixed brain sections for immunohistochemistry:

  • Mouse substantia nigra pars compacta

  • Mouse piriform cortex

  • Rat temporal cortex

When selecting samples, researchers should consider that SEZ6 expression is particularly high in the developing and postnatal forebrain, with significant expression maintained in the adult mouse cortex, hippocampus, striatum, olfactory tubercule, retina, and spinal cord .

How can SEZ6 antibodies distinguish between transmembrane and secreted SEZ6 isoforms?

SEZ6 exists in three splice isoforms: two transmembrane variants and one secreted form, each with distinct functional roles in neuronal development. The technical challenge in distinguishing these isoforms requires careful antibody selection and experimental design.

When designing experiments to differentiate between SEZ6 isoforms, researchers should consider:

• Epitope selection: Antibodies targeting the extracellular domain (like Anti-SEZ6 extracellular Antibody #ANR-206) will recognize both the transmembrane and secreted forms, while those targeting intracellular regions would detect only the transmembrane variants .

• Size differentiation: Western blot analysis can distinguish isoforms based on molecular weight differences. The observed band size of approximately 170 kDa for the full-length protein versus smaller bands for secreted forms can provide insight into isoform expression patterns .

• Subcellular localization: Immunohistochemical analysis can reveal whether SEZ6 is predominantly localized to the somatodendritic compartment (transmembrane form) or distributed in the extracellular space (secreted form) .

• BACE1 cleavage analysis: Since BACE1 cleaves the transmembrane form of SEZ6 to produce a shed ectodomain similar to the secreted SEZ6 isoform, co-immunoprecipitation experiments with BACE1 or inhibitor studies can help elucidate the processing mechanisms and distinguish between primary secreted forms and cleaved products .

What methodological approaches can optimize SEZ6 antibody use in studies of dendrite morphology?

Given SEZ6's critical role in dendrite development and branching, optimizing antibody-based techniques for morphological studies requires specific methodological considerations:

• Co-immunostaining protocol:

  • Combine Anti-SEZ6 antibody (1:300 dilution) with neuron-specific markers (e.g., MAP2 for dendrites, synaptophysin for presynaptic terminals)

  • Use confocal microscopy with z-stack acquisition to capture the three-dimensional dendritic arbor

  • Apply appropriate image analysis software for dendrite tracing and quantification

• Time-course analysis:

  • Implement time-course experiments during neuronal development to track SEZ6 expression in relation to dendrite elaboration

  • At each time point, process samples identically for consistent antibody penetration and signal detection

  • Correlate SEZ6 expression levels with morphological parameters (dendrite number, branch points, spine density)

• SEZ6 knockdown/overexpression validation:

  • Utilize the antibody to confirm altered SEZ6 expression in genetic manipulation studies

  • For knockout validation, ensure the antibody demonstrates absence of signal in SEZ6 KO tissues

  • When overexpressing specific isoforms, verify isoform-specific detection using appropriate antibodies

• High-resolution imaging considerations:

  • For detailed spine morphology analysis, super-resolution microscopy techniques may require modified antibody concentrations

  • Optimize fixation protocols to preserve delicate dendritic structures while maintaining antibody epitope accessibility

How can SEZ6 antibodies be applied in neuroendocrine tumor research?

Recent research has identified SEZ6 as a promising target for antibody-drug conjugate (ADC) therapy due to its abundant expression on neuroendocrine tumors, including small-cell lung cancer and small cell carcinoma of the ovary . Researchers investigating SEZ6's potential in oncology should consider the following methodological approaches:

• Expression profiling protocol:

  • Utilize Anti-SEZ6 antibodies in tissue microarrays containing various neuroendocrine tumors to quantify expression levels

  • Compare expression with normal tissue controls to confirm tumor specificity

  • Correlate expression levels with clinical parameters and patient outcomes

• Internalization kinetics assessment:

  • Since rapid internalization upon antibody binding makes SEZ6 a promising ADC target, develop pulse-chase experiments with fluorescently-labeled Anti-SEZ6 antibodies

  • Quantify internalization rates under various conditions using flow cytometry or confocal microscopy

  • Determine optimal antibody characteristics that promote efficient internalization

• ADC development considerations:

  • Evaluate various Anti-SEZ6 antibody clones for their binding affinity, specificity, and internalization efficiency

  • Test antibody-toxin conjugates for selective cytotoxicity against SEZ6-expressing tumor cells

  • Validate antibody specificity using SEZ6 blocking peptides to confirm binding selectivity

• Cross-reactivity assessment:

  • For preclinical development, confirm species cross-reactivity of SEZ6 antibodies (human, mouse, rat) to enable appropriate animal model studies

  • Establish standardized protocols for evaluating off-target binding using immunohistochemistry across a panel of normal tissues

What controls are essential when using SEZ6 antibodies in neurodevelopmental disorder research?

Given SEZ6's association with neurological conditions including autism spectrum disorder, epilepsy, intellectual disability, and schizophrenia , rigorous controls are necessary when investigating its role in these disorders:

• Specificity validation protocol:

  • Always include pre-absorption controls with specific blocking peptides (e.g., SEZ6 extracellular Blocking Peptide BLP-NR206)

  • Compare staining patterns between affected and control tissues processed simultaneously

  • For Western blot applications, include positive control samples with known SEZ6 expression

• Sample preparation standardization:

  • Standardize tissue collection, fixation times, and processing methods across patient and control samples

  • Document post-mortem intervals when using human tissue, as this may affect protein degradation

  • Match cases and controls for age, sex, and other relevant demographic factors

• Quantification methodology:

  • Implement blinded assessment of SEZ6 immunoreactivity

  • Utilize automated image analysis algorithms to reduce subjective interpretation

  • Include internal reference standards in each experimental batch to normalize between experiments

• Genetic variation considerations:

  • When studying patient populations with SEZ6 mutations, verify that the antibody's epitope is not affected by the specific genetic variations

  • Consider developing mutation-specific antibodies for certain research questions

  • Correlate antibody-detected protein levels with mRNA expression data to confirm findings

What technical challenges exist in detecting SEZ6 in cerebrospinal fluid for biomarker studies?

With elevated levels of SEZ6 observed in the cerebrospinal fluid (CSF) of patients with psychiatric disorders , developing robust detection methods presents unique challenges:

• Sensitivity optimization:

  • Adapt ELISA protocols similar to those developed for detecting antibodies to squalene , focusing on:

    • Optimal plate selection (polystyrene plates that provide low background and high specific binding)

    • Appropriate blocking agents (BSA rather than serum, which may contain interfering lipoproteins)

    • Signal amplification strategies for detecting low-abundance SEZ6

• Sample handling protocol:

  • Standardize CSF collection, processing, and storage conditions

  • Document factors that may influence SEZ6 levels (medication status, time of collection)

  • Include quality control samples in each assay run to monitor inter-assay variability

• Isoform discrimination strategy:

  • Develop sandwich ELISA approaches using antibody pairs targeting different SEZ6 epitopes

  • Design assays capable of distinguishing between full-length and cleaved forms of SEZ6 in CSF

  • Validate findings with orthogonal methods such as Western blotting or mass spectrometry

• Reference range establishment:

  • Collect sufficient control samples to establish normal reference ranges across different age groups

  • Account for potential confounding factors such as sex, age, and comorbidities

  • Implement statistical approaches to determine clinically significant cutoff values