SEPT6 Antibody

What is SEPT6 and why is it significant in cellular research?

SEPT6 belongs to the septins family of GTP-binding proteins that function as regulatable scaffolds for the recruitment of other proteins. These proteins are involved in critical cellular processes including membrane dynamics, vesicle trafficking, and apoptosis . Significant research interest in SEPT6 stems from its role in regulating neuronal cytoarchitecture, particularly in dendritic outgrowth and branching . SEPT6 begins significant expression at developmental stage 4 (dendritic outgrowth) in neurons, where it forms clusters at branch points of developing dendrites, suggesting its importance in neuronal morphogenesis . This temporal expression pattern makes SEPT6 a valuable marker for studying neuronal development and maturation.

What applications are SEPT6 antibodies validated for in research?

SEPT6 antibodies have been validated for multiple experimental applications as detailed below:

The antibody has shown reactivity with human, mouse, and rat samples, making it suitable for comparative studies across these species .

How can researchers verify SEPT6 antibody specificity?

Antibody specificity is crucial for accurate interpretation of experimental results. A rigorous specificity verification approach, as demonstrated in published research, includes:

• Immunoblot analysis: Using rat forebrain homogenate, a specific SEPT6 antibody should detect a predominant band at approximately 50 kDa, corresponding to the expected molecular weight of SEPT6 .

• Antigen competition assay: When the specific SEPT6 C-terminal peptide is added to the buffer, immunoblot signals at the 50 kDa position should weaken in a dose-dependent manner .

• Cross-reactivity testing: Testing with peptides from related septins (e.g., SEPT11) should not affect the 50 kDa SEPT6 band, confirming specificity .

• Gene knockout validation: Western blot analysis using brain tissue from SEPT6-deficient mice alongside wild-type controls provides definitive confirmation of antibody specificity .

What is the molecular weight of SEPT6 and how does this impact experimental design?

SEPT6 has a calculated molecular weight of approximately 50 kDa (434 amino acids) . This corresponds well with the observed molecular weight in experimental systems, as consistently detected at 50 kDa in Western blot analyses . The similarity in size to other septin family members (e.g., SEPT11 at 425 amino acids) necessitates careful antibody validation to ensure specificity . When designing experimental protocols, researchers should consider:

• Using appropriate percentage polyacrylamide gels (typically 10-12%) that provide optimal resolution in the 50 kDa range

• Including molecular weight markers that clearly demarcate the 50 kDa region

• Employing longer run times to better separate SEPT6 from similarly sized proteins

• Implementing controls to distinguish SEPT6 from other septins that may be present in the sample

What are the optimal conditions for Western blot detection of SEPT6?

For optimal Western blot detection of SEPT6, researchers should consider the following methodological details:

What antigen retrieval methods are recommended for SEPT6 immunohistochemistry?

For optimal immunohistochemical detection of SEPT6 in tissue samples, specific antigen retrieval methods have been validated:

• Primary recommendation: Antigen retrieval with TE buffer pH 9.0 has been suggested as the preferred method for SEPT6 detection in tissues like human cervical cancer tissue .

• Alternative method: Citrate buffer pH 6.0 can be used as an alternative antigen retrieval approach .

The choice between these methods may depend on specific tissue types and fixation protocols. It is advisable to compare both methods when establishing a new immunohistochemistry protocol for SEPT6 detection in unfamiliar tissue types.

How does SEPT6 expression change during neuronal development, and how should experiments be designed to capture these changes?

SEPT6 shows distinct developmental expression patterns in neurons that should inform experimental design:

• Temporal expression profile:

  • Very low expression until stage 3 (axonal outgrowth)

  • Significant expression begins at stage 4 (outgrowth of dendrites)

  • Continues expression through maturation (stage 5)

• Spatial localization changes:

  • Stage 4: SEPT6 clusters positioned at branch points of developing dendrites

  • Stage 5 (maturing and mature neurons): SEPT6 clusters positioned at the base of filopodia, spines, and pre-synaptic boutons

• Experimental design recommendations:

  • Include multiple time points capturing stages 3-5 of neuronal development

  • Utilize co-labeling with cytoskeletal markers (e.g., microtubules) to assess SEPT6 localization

  • Employ high-resolution imaging to visualize the tiny ring structures (~0.5μm diameter) formed by SEPT6

  • Include detergent extraction experiments to determine SEPT6 association with cellular structures (SEPT6 is not a post-synaptic density protein)

What controls should be included when using SEPT6 antibodies in gene disruption studies?

When utilizing SEPT6 antibodies in gene disruption studies, several critical controls should be implemented:

• Genotype verification: PCR-based genotyping of genomic DNA should be performed to confirm the genetic status (wild-type, heterozygous, or knockout) .

• Protein expression validation: Western blot analysis using anti-SEPT6 antibodies (recognizing epitopes within the carboxyl-terminal region) should confirm the absence of SEPT6 protein in knockout samples .

• Transcription confirmation: RT-PCR of total RNA (using primers covering multiple exons) should verify the disruption at the transcript level .

• Cellular phenotype assessment: As SEPT6 affects dendritic arborization, morphological analysis of neurons should be performed to confirm functional consequences of gene disruption .

• Compensatory mechanism evaluation: Analysis of other septin family members (particularly SEPT4) should be conducted to assess potential compensatory upregulation .

How can researchers distinguish between SEPT6 and other closely related septin family members?

Distinguishing SEPT6 from other septin family members requires careful methodological approaches:

• Antibody specificity: Select antibodies raised against unique regions of SEPT6. The C-terminal region has been successfully used to generate specific antibodies . A rigorous approach involves:

  • Comparing immunogen sequences with other septins for uniqueness

  • Testing cross-reactivity with recombinant septin proteins

  • Performing antigen competition assays with peptides from SEPT6 and related septins (e.g., SEPT11)

• Molecular weight considerations: While many septins have similar molecular weights (SEPT6: 434 aa, 50 kDa; SEPT11: 425 aa), subtle size differences can be leveraged using high-resolution SDS-PAGE .

• Expression pattern analysis: Different septins have distinct temporal and spatial expression patterns that can aid differentiation. For example, SEPT6 shows significant expression beginning at neuronal developmental stage 4, while other septins may show different patterns .

• Genetic models: Utilizing SEPT6 knockout/knockdown models as negative controls provides definitive confirmation of antibody specificity .

What are the methodological approaches for studying SEPT6's role in dendritic arborization?

Based on published research, several methodological approaches are effective for investigating SEPT6's function in dendritic branching:

• RNAi experiments: RNA interference has been successfully used to demonstrate that SEPT6 regulates dendritic arborization . This approach requires:

  • Design of specific siRNAs targeting SEPT6

  • Appropriate transfection methods for neuronal cultures

  • Quantitative assessment of knockdown efficiency

  • Morphological analysis of dendritic branching patterns

• High-resolution microscopy: Since SEPT6 forms tiny rings (~0.5μm diameter) at dendritic branch points, super-resolution imaging techniques may be required for detailed localization studies .

• Co-localization studies: SEPT6 interacts with the cytoskeleton, so co-labeling with microtubule markers can reveal functional associations during dendritic development .

• Live-cell imaging: For dynamic studies of SEPT6 during dendrite formation, fluorescently tagged SEPT6 constructs can be employed in time-lapse imaging experiments.

• Genetic models: SEPT6 knockout mice provide powerful tools for studying the consequences of SEPT6 loss on dendritic arborization in vivo .

How should researchers interpret subcellular SEPT6 localization in relation to neuronal function?

Interpreting SEPT6 subcellular localization requires understanding its changing patterns during neuronal development:

• Developmental stage correlation:

  • Stage 4 (dendritic outgrowth): SEPT6 clusters at branch points of developing dendrites suggest a role in branch formation

  • Stage 5 (maturation): SEPT6 clusters at the base of filopodia, spines, and pre-synaptic boutons indicate roles in synaptic organization

• Cytoskeletal association: SEPT6 forms rings on microtubule fibers, suggesting a structural role in organizing the cytoskeleton that supports dendritic branches .

• Membrane structures: SEPT6 is not a post-synaptic density (PSD) protein, as demonstrated by detergent extraction experiments . This indicates it may function in membrane organization rather than as a core component of the synaptic machinery.

• Functional implications: The positioning of SEPT6 at branch points and the base of dendritic protrusions suggests it may function as a scaffold for other proteins involved in establishing and maintaining these structures .

What are common technical challenges in SEPT6 antibody applications and their solutions?

Researchers may encounter several technical challenges when working with SEPT6 antibodies:

• Cross-reactivity with other septins:

  • Challenge: Due to sequence homology between septin family members, antibodies may detect multiple septins.

  • Solution: Perform antigen competition assays with peptides from different septins. A specific SEPT6 antibody signal should be blocked by SEPT6 peptides but not by peptides from other septins (e.g., SEPT11) .

• Variable signal intensity:

  • Challenge: SEPT6 expression varies by developmental stage and tissue type.

  • Solution: Optimize protein loading and antibody concentration for each experimental system. For neuronal studies, consider the developmental stage, as SEPT6 expression is very low until stage 4 .

• Background in immunohistochemistry:

  • Challenge: Non-specific binding in tissue sections.

  • Solution: Optimize antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0) and implement thorough blocking steps .

• Storage-related antibody performance decline:

  • Challenge: Decreased sensitivity over time.

  • Solution: Store antibody at -20°C in aliquots to avoid freeze-thaw cycles. The antibody is typically stable for one year after shipment when properly stored .

How can researchers validate functional findings from SEPT6 knockdown/knockout studies?

Validation of functional findings from SEPT6 manipulation studies should incorporate multiple approaches:

• Multiple knockdown/knockout strategies: Utilize different methods (siRNA, CRISPR-Cas9, gene targeting) to ensure the observed phenotype is not due to off-target effects .

• Rescue experiments: Re-expressing SEPT6 in knockout/knockdown models should reverse the observed phenotype if it is specifically due to SEPT6 loss.

• Dose-dependency assessment: Where possible, create hypomorphic as well as null alleles to establish correlation between SEPT6 levels and phenotype severity.

• Temporal control: Implementing inducible knockdown/knockout systems can distinguish between developmental and maintenance roles of SEPT6.

• Cross-species validation: Confirming findings across multiple model systems (e.g., mouse and rat) strengthens confidence in the results .

• Phenotypic specificity: Compare the phenotype of SEPT6 disruption with disruption of other septins (e.g., SEPT4) to identify septin-specific versus general septin family functions .

What are the implications of SEPT6 knockout studies for understanding septin biology?

SEPT6 knockout studies have revealed several important insights and raised questions about septin biology:

• Developmental redundancy: Despite SEPT6's role in dendritic arborization in cellular models, SEPT6-deficient mice were born with predicted Mendelian frequencies in both sexes, developed without growth retardation, were fertile, and had normal lifespans . This suggests potential compensatory mechanisms within the septin family.

• Functional compensation: The absence of gross abnormalities in SEPT6 knockout mice indicates that other septins may compensate for SEPT6 loss during development . This points to functional redundancy that should be considered when designing experiments targeting septins.

• Context-dependent functions: The disconnect between cellular phenotypes (altered dendritic arborization) and whole-organism development suggests SEPT6 functions may be more critical in specific contexts or under particular stresses not encountered in standard laboratory conditions .

• Experimental design implications: These findings highlight the importance of studying septin functions at multiple levels—molecular, cellular, and organismal—and the need to consider compensatory mechanisms when interpreting results .

How can researchers best approach studying SEPT6 in the context of disease models?

When investigating SEPT6 in disease models, researchers should consider these methodological approaches:

• Expression correlation studies: Analyze SEPT6 expression levels in disease-relevant tissues compared to healthy controls. SEPT6 antibodies have been validated for detection in human, mouse, and rat samples .

• Localization alterations: Examine changes in SEPT6 subcellular localization in disease states, particularly in neurological disorders where dendritic architecture may be compromised .

• Genetic association studies: Investigate whether SEPT6 genetic variants correlate with disease susceptibility or progression, particularly in neurodevelopmental disorders.

• Conditional knockout models: Generate tissue-specific or inducible SEPT6 knockout models to avoid potential developmental compensation and better model adult-onset disorders .

• Combined septin targeting: Given the potential redundancy among septins, consider approaches that target multiple septin family members simultaneously or that analyze the entire septin interactome in disease contexts .

• Therapeutic targeting assessment: Evaluate whether modulation of SEPT6 function or expression affects disease progression in relevant model systems.

The lack of gross abnormalities in SEPT6 knockout mice suggests that therapeutic targeting of SEPT6 might have limited side effects, making it potentially interesting for therapeutic development if disease-specific functions are identified .