SEZ6L2 Antibody, HRP conjugated

Basic Research Questions

• What is SEZ6L2 and why is it important to study?

  SEZ6L2 (seizure related 6 homolog like 2) is a type 1 transmembrane protein predominantly expressed in the brain. In humans, the canonical protein has 910 amino acid residues with a molecular mass of 97.6 kDa, though glycosylation can increase this to 150-170 kDa in observed experiments . SEZ6L2 belongs to the SEZ6 protein family, which includes SEZ6, SEZ6L, and SEZ6L2.

  The protein is significant because it:

  • Contributes to specialized endoplasmic reticulum functions in neurons

  • Is primarily localized to the ER and cell membrane

  • Undergoes alternative splicing to produce 6 different isoforms

  • Features post-translational modifications, particularly O-glycosylation

  • Functions as a complement regulator by inhibiting C3 convertases and promoting C3b degradation

  • Plays roles in both neurological processes and cancer progression

  Studying SEZ6L2 is critical for understanding brain function, neurodevelopmental disorders, and various cancer pathologies where it shows aberrant expression.

• What experimental applications are most suitable for HRP-conjugated SEZ6L2 antibodies?

  HRP-conjugated SEZ6L2 antibodies are particularly valuable for specific applications:

  Application	Suitability	Key Considerations
  Western Blot (WB)	Excellent	Primary detection method; eliminates need for secondary antibody
  ELISA	Excellent	Direct detection with enhanced sensitivity
  Immunohistochemistry (IHC)	Good	May require optimization of antigen retrieval; provides direct visualization
  Flow Cytometry (FCM)	Good	Useful for cell surface detection of SEZ6L2

  HRP conjugation provides direct enzymatic detection capability, eliminating secondary antibody steps and potentially reducing background or cross-reactivity issues. This makes these antibodies particularly valuable for techniques requiring high sensitivity and specificity, such as detecting low abundance SEZ6L2 in complex samples or tissues .

• How can I validate the specificity of a SEZ6L2 antibody?

  Proper validation of SEZ6L2 antibodies requires multiple approaches:

  • Positive controls: Use mouse or rat brain tissue lysates, where SEZ6L2 is highly expressed

  • Western blot analysis: Verify a band at approximately 98-100 kDa (unglycosylated) or 150-170 kDa (glycosylated form)

  • Knockout/knockdown validation: Compare SEZ6L2 knockdown samples to confirm antibody specificity

  • Cross-reactivity testing: Ensure the antibody doesn't react with other SEZ6 family members (SEZ6 and SEZ6L)

  • Peptide competition: Block antibody binding with a specific peptide blocking solution corresponding to the immunogen sequence

  • Application-specific validation: For IHC, verify correct subcellular localization patterns in brain tissue sections

  A comprehensive validation strategy employing multiple techniques provides the strongest evidence for antibody specificity and suitability for your specific research applications.

• What is the significance of HRP conjugation for SEZ6L2 antibody applications?

  Horseradish peroxidase (HRP) conjugation offers several distinct advantages in SEZ6L2 research:

  • Direct detection: Eliminates the need for secondary antibodies, reducing experimental variability and potential cross-reactivity issues

  • Enhanced sensitivity: HRP amplifies signal through enzymatic activity with chromogenic or chemiluminescent substrates, enabling detection of low abundance SEZ6L2 in complex samples

  • Reduced background: Fewer incubation steps minimize non-specific binding opportunities

  • Time efficiency: Simplifies workflows by eliminating secondary antibody incubation and washing steps

  • Quantification potential: Offers precise signal quantification when used with appropriate substrates in applications like ELISA

  • Versatility: Compatible with various detection methods (chromogenic, chemiluminescent, fluorescent) depending on substrate choice

  The HRP enzyme catalyzes the oxidation of substrates in the presence of hydrogen peroxide, generating detectable signals particularly valuable for precise localization of SEZ6L2 in tissue sections or quantitative analysis in biochemical assays.

• What are the optimal storage conditions for HRP-conjugated SEZ6L2 antibodies?

  Proper storage is crucial for maintaining HRP-conjugated antibody performance:

  • Light protection: Store in light-protected vials or cover with aluminum foil to prevent photodegradation of the HRP enzyme

  • Temperature: Maintain at 4°C for short-term storage (up to 12 months)

  • Long-term storage: For extended storage (up to 24 months), dilute with up to 50% glycerol and store at -20°C to -80°C

  • Avoid freeze-thaw cycles: Repeated freezing and thawing significantly compromises both enzyme activity and antibody binding capability

  • Aliquoting: For antibodies requiring freezing, create single-use aliquots to prevent freeze-thaw damage

  • Buffer considerations: Store in manufacturer-recommended buffer systems (typically PBS with stabilizers)

  Following these guidelines ensures maximum retention of both antibody specificity and HRP enzymatic activity, critical for consistent experimental results.

Advanced Research Applications

• How can Western blot protocols be optimized for SEZ6L2 detection?

  Optimizing Western blot for SEZ6L2 requires attention to several technical aspects:

  • Sample preparation: For brain tissue samples, use buffers containing phosphatase and protease inhibitors to prevent protein degradation

  • Protein loading: Load 20-40 μg of total protein for brain tissue lysates; may require higher amounts for tissues with lower expression

  • Gel percentage: Use 8-10% SDS-PAGE gels to effectively resolve the high molecular weight SEZ6L2 (~98-170 kDa)

  • Transfer conditions: Perform wet transfer at 30V overnight at 4°C for complete transfer of high molecular weight SEZ6L2

  • Blocking conditions: 5% non-fat dry milk in TBST is generally effective; for phosphorylation studies, use 5% BSA

  • Antibody dilution: For HRP-conjugated SEZ6L2 antibodies, a 1:1000-1:4000 dilution range is typically effective

  • Washing stringency: Include 0.05-0.1% Tween-20 in wash buffers to reduce background while preserving specific signal

  • Detection method: Use enhanced chemiluminescence (ECL) with sensitive substrates for optimal visualization

  • Special considerations: Account for glycosylation, which can shift SEZ6L2 bands to approximately 150-170 kDa

  Western blots should be conducted under reducing conditions using appropriate immunoblot buffer systems for optimal results .

• What are the key considerations when studying SEZ6L2 expression in different neural tissues?

  Neural tissue analysis of SEZ6L2 requires attention to region-specific and developmental factors:

  • Expression patterns: SEZ6L2 shows variable expression across brain regions, with particularly high expression in the hippocampus

  • Developmental timing: Consider ontogenetic expression patterns, as SEZ6L2 shows differential expression during development (e.g., 15 d.p.c. embryos show specific expression in developing brain, spinal cord, trigeminal ganglia, and retina)

  • Cellular localization: Use confocal microscopy with co-localization markers to distinguish between ER, membrane, and other subcellular compartments

  • Tissue preparation: For IHC, perfusion-fixed frozen sections yield superior results compared to paraffin embedding for SEZ6L2 detection

  • Signal amplification: For regions with lower expression, consider tyramide signal amplification systems with HRP-conjugated antibodies

  • Counterstaining: Use neuronal markers (NeuN, MAP2) to identify specific cell populations expressing SEZ6L2

  • Species considerations: Note potential differences between human, mouse, and rat SEZ6L2 expression patterns when designing experiments

  Comparative analysis across brain regions should include standardized internal controls to account for regional differences in protein extraction efficiency and background autofluorescence.

• How does SEZ6L2 glycosylation affect antibody binding and experimental outcomes?

  SEZ6L2 undergoes extensive glycosylation that significantly impacts experimental approaches:

  • Molecular weight variation: Glycosylation increases observed molecular weight from the predicted 97.6 kDa to approximately 150-170 kDa in Western blots

  • Epitope masking: Heavy glycosylation may obscure antibody binding sites, particularly in native protein conformations

  • Antibody selection strategy: Choose antibodies targeting regions less likely to be affected by glycosylation, such as those directed against middle regions or cytoplasmic domains

  • Deglycosylation considerations: For some applications, enzymatic deglycosylation (PNGase F, O-glycosidase) before analysis may improve detection consistency

  • Tissue-specific glycosylation: Different tissues may exhibit variable glycosylation patterns, affecting antibody binding affinity

  • Detection method comparison: Non-denaturing methods (ELISA, IP) may be more affected by glycosylation than denaturing methods (Western blot)

  Researchers should verify which form of SEZ6L2 (glycosylated vs. non-glycosylated) their specific antibody recognizes best to ensure appropriate experimental design and interpretation of results.

• What experimental approaches can reveal SEZ6L2's role in complement regulation?

  Investigating SEZ6L2's role in complement regulation requires specialized methods:

  • Hemolytic assays: Both classical and alternative pathway hemolytic assays can measure SEZ6L2's inhibitory effect on complement-mediated lysis of erythrocytes

  • Factor I cofactor activity analysis: Assess SEZ6L2's ability to promote C3b cleavage using purified proteins and Western blot detection

  • C3 convertase dissociation studies: Measure how SEZ6L2 accelerates the decay of C3 convertases using surface plasmon resonance or other binding assays

  • Domain mapping: Use truncated versions of SEZ6L2 (e.g., Sez6L2-MH that lacks transmembrane and cytoplasmic regions) to identify which domains are responsible for complement regulatory functions

  • Functional rescue experiments: In knockdown models, reintroduce wild-type or mutant SEZ6L2 to determine which domains are necessary for complement regulation

  • Cell-based assays: Assess protection from complement-mediated damage in cells expressing different levels of SEZ6L2

  Experimental designs should include appropriate positive controls such as established complement regulators (Factor H, DAF) to benchmark SEZ6L2's efficacy in complement inhibition .

• How can SEZ6L2 knockdown experiments be optimized for studying apoptotic pathways?

  Knockdown studies of SEZ6L2 to investigate apoptosis require careful methodological considerations:

  • Knockdown validation: Confirm effective SEZ6L2 knockdown using both mRNA (qRT-PCR) and protein (Western blot) analysis

  • Delivery systems: Lentivirus-based shRNA systems show high efficiency for stable SEZ6L2 knockdown in cancer cell lines

  • Selection methods: Use puromycin selection to establish stable cell lines with SEZ6L2 knockdown

  • Multiple targeting sequences: Employ at least two different shRNA sequences targeting SEZ6L2 to rule out off-target effects

  • Apoptosis detection methods: Combine flow cytometry (Annexin V/PI staining) with Western blot analysis of apoptotic markers (cleaved caspase 3, cleaved caspase 9)

  • Pathway dissection: Distinguish between intrinsic (mitochondrial) and extrinsic apoptotic pathways by examining caspase 8 (extrinsic) versus caspase 9 (intrinsic) activation

  • In vivo validation: Confirm in vitro findings using xenograft models with TUNEL assays to detect apoptotic cells in tumor tissues

  SEZ6L2 knockdown significantly promotes apoptosis in colorectal cancer cells through activation of caspase 3 and caspase 9, suggesting involvement in the intrinsic apoptotic pathway. This approach can be adapted to study SEZ6L2's role in various cancer types .

Technical Methodology

• What are the recommended blocking conditions for SEZ6L2 antibody in immunohistochemistry?

  Optimal blocking for SEZ6L2 immunohistochemistry depends on the tissue and detection system:

  Tissue Type	Recommended Blocking	Incubation	Notes
  Brain frozen sections	5-10% normal serum from secondary antibody host species	1 hour, RT	For HRP-conjugated antibodies, use serum from same species as tissue
  FFPE tissue	0.3% H₂O₂ followed by 2-5% BSA	10 min (H₂O₂), 1 hour (BSA)	H₂O₂ block is critical to reduce endogenous peroxidase activity
  Cultured cells	1-5% BSA or 5% normal serum	30-60 min, RT	Include 0.1-0.3% Triton X-100 for permeabilization

  For brain tissues, where SEZ6L2 is highly expressed, additional considerations include:

  • Use 0.3% H₂O₂ in methanol for endogenous peroxidase quenching prior to primary antibody incubation

  • Consider adding 0.1% Tween-20 to blocking solutions to reduce background

  • For highly autofluorescent tissues, include 0.1-0.3% Sudan Black B in 70% ethanol after antibody incubation

  • Overnight incubation at 4°C with primary antibody typically yields optimal signal-to-noise ratio

• How do different fixation methods affect SEZ6L2 antibody performance in IHC?

  Fixation significantly impacts SEZ6L2 detection in immunohistochemistry:

  Fixation Method	Impact on SEZ6L2 Detection	Recommended Protocol
  Perfusion fixation	Superior preservation of antigenic sites	4% PFA perfusion followed by post-fixation for 2-4 hours
  Immersion fixation	Adequate for embryonic tissues	4% PFA for 24-48 hours at 4°C
  Methanol fixation	Preserves some epitopes but may disrupt membrane localization	-20°C methanol for 10 minutes
  FFPE processing	May mask epitopes, requiring antigen retrieval	Citrate buffer (pH 6.0) heat-induced retrieval

  For optimal SEZ6L2 detection in adult brain tissue, perfusion-fixed frozen sections yield superior results compared to paraffin embedding . Cryopreservation in 30% sucrose after fixation helps maintain tissue integrity during freezing. When using HRP-conjugated antibodies, shorter fixation times may reduce background from fixative-induced autofluorescence or peroxidase activity.

• What is the recommended dilution range for HRP-conjugated SEZ6L2 antibodies in various applications?

  Optimal dilutions vary by application and specific antibody formulation:

  Application	Recommended Dilution Range	Starting Concentration	Optimization Strategy
  Western Blot	1:1000-1:4000	0.5 mg/ml stock	Begin with 1:1000 and titrate for optimal signal-to-noise
  IHC-Frozen sections	1:100-1:500	0.5 mg/ml stock	Start with 1:200 and adjust based on background levels
  IHC-Paraffin sections	1:50-1:200	0.5 mg/ml stock	Higher concentrations often needed; include thorough antigen retrieval
  ELISA	1:500-1:5000	0.5 mg/ml stock	Perform checkerboard titration against known positive samples
  Flow Cytometry	1:100-1:400	0.5 mg/ml stock	Higher concentrations than WB typically required

  For all applications, include both positive and negative controls at each dilution during optimization. The concentration of 0.5 mg/ml is typical for commercially available antibodies , but always refer to manufacturer specifications for the specific antibody being used.

• What control samples should be included when studying SEZ6L2 in cancer research?

  Comprehensive controls are essential for SEZ6L2 cancer studies:

  • Positive tissue controls: Brain tissue (particularly hippocampus) serves as a reliable positive control for SEZ6L2 expression

  • Normal adjacent tissue: For cancer studies, paired normal tissue from the same patient provides critical baseline comparison

  • Expression controls: Include cell lines with known SEZ6L2 expression levels (HCT116 and HT29 show high expression in colorectal cancer studies)

  • Knockdown validation: Cells with confirmed SEZ6L2 knockdown serve as specificity controls for antibody detection

  • Isotype controls: Include matching isotype antibodies to assess non-specific binding, particularly important in IHC and flow cytometry

  • Technical controls: For Western blot, include loading controls (β-actin, GAPDH) and molecular weight markers

  In colorectal cancer research specifically, human intestinal epithelial cells (HIEC) have been used as normal controls for comparison with cancer cell lines . Tissue microarrays containing multiple cancer types can help establish expression patterns across different malignancies.

• How can I troubleshoot high background when using HRP-conjugated SEZ6L2 antibodies?

  High background with HRP-conjugated antibodies can be resolved through systematic approaches:

  Problem	Potential Causes	Solutions
  Diffuse background	Insufficient blocking	Increase blocking time and concentration; try different blocking agents (BSA, normal serum, commercial blockers)
  Speckled background	Antibody aggregation	Centrifuge antibody before use; filter through 0.22μm filter; ensure proper storage conditions
  Edge effects in IHC	Drying during incubation	Maintain humidity during incubations; use larger volumes of antibody solution
  High background in Western blot	Insufficient washing	Increase number and duration of washes; add 0.05-0.1% Tween-20 to wash buffers
  Non-specific tissue binding	Endogenous biotin or peroxidase	Include avidin/biotin blocking step; use 0.3% H₂O₂ pretreatment
  Signal in negative controls	Antibody concentration too high	Further dilute primary antibody; reduce incubation time

  For persistent problems, consider:

  • Using alternative detection systems (e.g., biotin-free detection)

  • Employing antigen retrieval optimization for IHC applications

  • Testing different membrane types for Western blot

  • Preparing fresh buffers and blocking solutions

  • Reducing substrate incubation time to minimize background development

Disease and Pathway Research

• How is SEZ6L2 involved in cancer progression and what experimental approaches best investigate this role?

  SEZ6L2 shows significant oncogenic potential through multiple mechanisms:

  • Expression analysis: SEZ6L2 is significantly upregulated in multiple cancer types, including colorectal cancer, lung cancer, hepatocellular carcinoma, and thyroid carcinoma

  • Prognostic correlation: Higher SEZ6L2 expression correlates with advanced disease stage (phase II-III vs. phase I) in colorectal cancer patients

  • Experimental approaches:

    1. Knockdown studies: Lentivirus-based shRNA targeting SEZ6L2 significantly inhibits cancer cell growth both in vitro and in vivo

    2. Proliferation assays: CCK-8 assays and colony formation assays demonstrate reduced growth capabilities in SEZ6L2-knockdown cells

    3. Apoptosis assessment: Flow cytometry and Western blot detection of apoptotic markers reveal SEZ6L2's anti-apoptotic function

    4. Xenograft models: In vivo tumor growth is significantly impaired with SEZ6L2 knockdown, with tumor volume reductions of 52-60%

    5. Pathway analysis: Western blotting for caspase activation (particularly caspase 3 and 9) reveals involvement in intrinsic apoptotic pathways

    6. TUNEL assays: In situ detection of apoptotic cells in tumor tissues confirms in vitro findings

  Notably, SEZ6L2 knockdown specifically promotes mitochondria-related apoptosis without significantly affecting cancer cell invasion capabilities . These findings suggest SEZ6L2 as a potential therapeutic target for cancer treatment.

• What is known about SEZ6L2's function in neurological processes and how can it be investigated?

  SEZ6L2's neurological functions are diverse and can be studied through specialized approaches:

  • Synaptic function: SEZ6L2 acts as a scaffolding protein linking GluR1 to adducin in AMPA receptors , suggesting a role in synaptic transmission

  • Structural effects: SEZ6 family members influence synapse numbers and dendritic morphology , suggesting developmental roles

  • Neurological disorders: SEZ6L2 has been linked to various neurological and psychiatric conditions

  • Investigation methods:

    1. Immunohistochemistry: Examine neuroanatomical distribution in specific brain regions such as hippocampus

    2. Developmental analysis: Study expression patterns during neurodevelopment (e.g., in embryonic brain, spinal cord, and retina)

    3. Co-localization studies: Determine subcellular distribution and protein interactions through confocal microscopy

    4. Functional assays: Electrophysiology to measure AMPA receptor function in presence/absence of SEZ6L2

    5. Behavioral assessments: Evaluate cognitive, motor, and social behaviors in animal models with altered SEZ6L2 expression

    6. Proteomic analysis: Identify binding partners through co-immunoprecipitation coupled with mass spectrometry

  The presence of SEZ6L2 in developing brain structures suggests important neurodevelopmental functions that may explain its association with neurological disorders when dysregulated .

• How does the molecular structure of SEZ6L2 relate to its diverse biological functions?

  SEZ6L2's structure contains specific domains relating to its various functions:

  • Transmembrane domain: Anchors the protein in the ER and cell membrane, positioning it for both intracellular and extracellular interactions

  • CUB domains: May mediate protein-protein interactions, particularly relevant for complement regulation

  • Sushi/SCR/CCP domains: Present in many complement regulators, likely responsible for SEZ6L2's complement inhibitory activity

  • Glycosylation sites: Multiple O-glycosylation modifications affect protein stability and interactions

  • Structure-function studies:

    1. Domain mapping: Truncated versions (e.g., Sez6L2-MH lacking transmembrane region) help identify functional domains

    2. Recombinant expression: Using mammalian expression systems to produce tagged versions for purification and functional studies

    3. Site-directed mutagenesis: Targeting specific residues to determine their importance for protein function

    4. Structural biology approaches: Crystallography or cryo-EM to determine three-dimensional structure

    5. Bioinformatic analysis: Sequence comparisons with other SEZ6 family members to identify conserved functional regions

  Understanding SEZ6L2's structural features provides insight into its multifunctional capabilities in different cellular contexts, from complement regulation to synaptic organization and cancer progression.

• What techniques are most effective for distinguishing between SEZ6L2 and other SEZ6 family members?

  Differentiating between closely related SEZ6 family proteins requires specific approaches:

  • Antibody selection: Choose antibodies verified for lack of cross-reactivity with other family members (SEZ6, SEZ6L)

  • Western blot differentiation: SEZ6L2 appears at ~98 kDa (unglycosylated) or 150-170 kDa (glycosylated), which may differ from other family members

  • Peptide competition: Use specific blocking peptides corresponding to unique regions of SEZ6L2

  • qRT-PCR primers: Design primers targeting unique regions of SEZ6L2 mRNA for expression analysis

  • RNA interference: Use targeted siRNA/shRNA with confirmed specificity for SEZ6L2

  • Immunoprecipitation: Pull down with specific antibodies followed by mass spectrometry can confirm identity

  • Immunofluorescence patterns: Compare subcellular localization patterns between family members

  In direct ELISA testing, high-quality antibodies show no cross-reactivity between SEZ6L2 and other family members like BSRP-B or BSRP-C , confirming the ability to distinguish these proteins despite their structural similarities.

• What is the current understanding of SEZ6L2's dual role in neurobiology and cancer pathology?

  SEZ6L2's involvement in both neurological function and cancer reveals interesting biological duality:

  • Neurological functions:

    • Contributes to specialized ER functions in neurons

    • Influences synapse formation and dendritic morphology

    • Acts as a scaffolding protein in AMPA receptors

    • Linked to neurodevelopmental disorders

  • Cancer pathology:

    • Significantly upregulated in multiple cancer types

    • Inhibits apoptosis through caspase-dependent mechanisms

    • Promotes tumor growth in vivo

    • Associated with advanced disease stages

  • Potential connecting mechanisms:

    1. Signaling pathway overlap: Common signaling pathways may be utilized differently in neurons versus cancer cells

    2. Alternative splicing: Different isoforms (6 reported) may have tissue-specific functions

    3. Post-translational modifications: Differential glycosylation patterns may alter function in different contexts

    4. Protein interactions: Different binding partners in neural versus cancer tissues

    5. Complement regulation: SEZ6L2's role in complement inhibition could impact both neural development and tumor immune evasion

  This dual functionality makes SEZ6L2 a particularly interesting target for both neurological research and cancer biology, with potential implications for therapeutic development in both fields.