SEC14L3 Antibody

What is SEC14L3 protein and what are its primary biological functions?

SEC14L3 is a probable hydrophobic ligand-binding protein that plays a role in the transport of hydrophobic ligands including tocopherol, squalene, and phospholipids . It shares significant homology with the Saccharomyces cerevisiae SEC14 protein, which functions as a phosphatidylinositol transfer protein essential for biogenesis of Golgi-derived transport vesicles . Recent studies have identified SEC14L3 as a component in non-canonical Wnt/Ca2+ signaling pathways, where it interacts with several key proteins involved in this signaling cascade .

The protein contains several functional domains, including CARL-TRIO, GOLD, and SEC14 domains, each contributing to specific protein-protein interactions and biochemical functions . The SEC14 domain appears particularly important for interactions with Dishevelled (Dvl) proteins, which are central mediators of Wnt signaling .

What applications are SEC14L3 antibodies validated for in research settings?

SEC14L3 antibodies have been validated for multiple research applications, with varying degrees of optimization across different experimental platforms:

These applications have been confirmed across multiple tissue types, including liver, lung, testis, and various cancer tissues . When implementing any of these techniques, preliminary titration experiments are recommended to determine optimal antibody concentrations for your specific experimental system.

How should researchers properly store and handle SEC14L3 antibodies?

SEC14L3 antibodies typically come in lyophilized form and require proper reconstitution and storage for optimal performance. The recommended protocol includes:

• Store lyophilized antibody at -20°C for up to one year from the date of receipt .

• Reconstitute by adding 0.2mL of distilled water to achieve a final concentration of approximately 500μg/mL .

• After reconstitution, store at 4°C for up to one month for active use .

• For long-term storage, aliquot the reconstituted antibody and store at -20°C for up to six months .

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and reduce binding efficiency .

Reconstitution buffers typically contain stabilizing agents such as trehalose (4mg), NaCl (0.9mg), and Na₂HPO₄ (0.2mg) per vial to maintain antibody integrity .

What antigen retrieval methods are optimal for SEC14L3 detection in fixed tissues?

Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has been demonstrated as effective for SEC14L3 detection in paraffin-embedded tissues . The validated protocol includes:

• Deparaffinize and rehydrate tissue sections following standard protocols.

• Perform heat-mediated antigen retrieval using EDTA buffer (pH 8.0) as the epitope retrieval solution .

• Block tissue sections with 10% goat serum to reduce non-specific binding .

• Incubate sections with anti-SEC14L3 antibody (recommended concentration: 2μg/mL) overnight at 4°C .

• Apply biotinylated secondary antibody (e.g., biotinylated goat anti-rabbit IgG for rabbit primary antibodies) and incubate for 30 minutes at 37°C .

• Develop using a Streptavidin-Biotin-Complex with DAB as the chromogen .

This protocol has been validated in multiple tissue types, including rat testis, human breast cancer, and human gastric cancer tissues .

What controls should be included when working with SEC14L3 antibodies?

Implementing appropriate controls is critical for interpreting results with SEC14L3 antibodies:

• Positive tissue controls: Lung and testis tissues have demonstrated consistent SEC14L3 expression and can serve as positive controls .

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • Tissues known to lack SEC14L3 expression

• Blocking peptide control: Using the immunogen peptide (positions R4-V400 of human SEC14L3) to pre-absorb the antibody can confirm specificity .

• Knockdown/knockout validation: When available, samples from SEC14L3 knockdown or knockout models provide the most stringent specificity control. Studies using morpholino oligonucleotides (MO) targeting SEC14L3 in zebrafish have shown specific phenotypes that can be rescued by re-expression of the protein .

How does SEC14L3 participate in Wnt signaling pathways and what experimental approaches can detect these interactions?

SEC14L3 has been identified as a component in non-canonical Wnt/Ca2+ signaling, forming protein complexes with Frizzled (Fz) receptors, Dishevelled (Dvl) proteins, and Phospholipase C (PLC) . These interactions can be studied through multiple approaches:

• Co-immunoprecipitation (Co-IP): SEC14L3 has been shown to associate with C-terminal regions of human Frizzled 5 (hFz5-CT) and rat Frizzled 2 (Rfz2-CT) in HEK293T cells . Researchers can immunoprecipitate with anti-Flag antibodies when using Flag-tagged SEC14L3 constructs.

• Direct binding assays: Direct protein-protein interactions can be demonstrated using purified proteins. GST-SEC14L3 and hFz5-CT/Rfz2-CT-Myc proteins expressed in E. coli and purified have confirmed direct binding in vitro .

• Complex formation analysis: Sequential immunoprecipitation approaches have demonstrated that hFz5-CT, mDvl2, and SEC14L3 can form ternary complexes . This involves:

  • Incubating purified hFz5-CT-Myc and GST-SEC14L3 proteins with mDvl2-Flag transfected cell lysates

  • Sequential pull-down using GST antibody (1st IP) and Flag antibody (2nd IP)

  • Detection of co-precipitated proteins using corresponding antibodies

• Domain mapping: Using deletion constructs of SEC14L3, researchers have identified that:

  • CARL-TRIO and GOLD domains mediate interaction with Rfz2-CT

  • SEC14 domain is necessary for interaction with Dvl2

What is the functional significance of SEC14L3 in embryonic development and how can this be studied?

SEC14L3 has demonstrated roles in embryonic development through its participation in calcium signaling pathways. Key experimental approaches include:

• Morpholino knockdown: Antisense morpholino oligonucleotides targeting SEC14L3 (sec14l3-MO1 and sec14l3-MO2) have been used to block its translation in zebrafish embryos, resulting in slower epiboly in a dose-dependent manner .

• CRISPR-Cas9 mutagenesis: Generation of SEC14L3 mutant lines provides a more stable genetic model for studying developmental phenotypes .

• Calcium signaling assays: Since SEC14L3 affects non-canonical Wnt/Ca2+ signaling, calcium imaging techniques can be employed to visualize altered calcium dynamics in SEC14L3-deficient embryos.

• Epistasis experiments: Determining whether SEC14L3 functions upstream or downstream of other Wnt pathway components can be achieved through rescue experiments combining SEC14L3 knockdown with overexpression of other pathway components.

How can researchers distinguish between the functions of different SEC14-like family proteins?

The SEC14-like family includes multiple members (SEC14L2, SEC14L3, etc.) with potentially overlapping functions. Experimental approaches to distinguish their specific roles include:

• Expression profiling: Different SEC14-like proteins show tissue-specific expression patterns. For example, human SEC14L2 is expressed in HEK293T and PC3 cells, while SEC14L3 shows different expression patterns .

• Domain-specific interactions: Mapping the protein-protein interactions of each family member can reveal functional differences. The SEC14 domain of SEC14L3 is required for interaction with Dvl2, while other domains mediate different interactions .

• Parallel knockdown/knockout studies: Comparing phenotypes resulting from deficiency of different family members can highlight unique functions.

• Rescue experiments: Testing whether one family member can functionally substitute for another provides insight into shared versus distinct functions.

What factors may affect SEC14L3 antibody performance and how can these be mitigated?

Several factors can impact SEC14L3 antibody performance in experimental applications:

• Epitope accessibility: The immunogen used for antibody production (positions R4-V400 of human SEC14L3) may have varying accessibility in different sample preparations . Optimizing fixation and antigen retrieval protocols is crucial.

• Cross-reactivity: While commercial antibodies claim no cross-reactivity with other proteins, validation in your specific experimental system is recommended .

• Antibody concentration: Titration experiments should be conducted to determine optimal concentrations for different applications. Starting recommendations are 1:500 for Western blot and 1:100 for immunohistochemistry .

• Sample preparation: Protein denaturation conditions for Western blot may affect epitope recognition. Testing both reducing and non-reducing conditions may be necessary.

• Signal amplification: For tissues with low SEC14L3 expression, signal amplification systems like Streptavidin-Biotin-Complex (SABC) with DAB as chromogen have proven effective .

What is the expected molecular weight pattern for SEC14L3 in Western blot analysis?

SEC14L3 protein typically appears at the following molecular weights:

• Observed molecular weight: 46-47 kDa

• Calculated molecular weight: 64 kDa

This discrepancy between observed and calculated molecular weights may result from:

• Post-translational modifications

• Protein processing

• Alternative splicing (alternative transcript variants have been observed for this gene)

• Gel migration characteristics

When performing Western blot, researchers should be aware of this potential size difference and include appropriate molecular weight markers.

How is SEC14L3 expression altered in disease states and what are the implications for using SEC14L3 antibodies in disease research?

SEC14L3 antibodies have been validated in several disease-relevant tissues:

• Cancer research: SEC14L3 has been detected in:

  • Human breast cancer tissue

  • Human gastric cancer tissue

  • Human lung cancer tissue

• Potential disease associations: Based on its role in Wnt signaling pathways, SEC14L3 may be implicated in diseases associated with dysregulated Wnt signaling, including:

  • Developmental disorders

  • Cancer progression

  • Inflammatory conditions

Future research directions might include:

• Comparative analysis of SEC14L3 expression in normal versus diseased tissues

• Correlation of expression levels with disease progression or prognosis

• Investigation of SEC14L3 as a potential therapeutic target or biomarker

What model systems are most appropriate for studying SEC14L3 function?

Based on available research, several model systems have been validated for SEC14L3 studies:

• Zebrafish embryos: Effective for developmental studies, with phenotypes observed upon SEC14L3 knockdown using morpholino oligonucleotides .

• Cell culture systems:

  • HEK293T cells: Suitable for protein interaction studies, although they primarily express SEC14L2 rather than SEC14L3

  • MCF7 cells: Express endogenous SEC14L2 and show interactions with DVL2

• Tissue samples:

  • Mouse liver and lung lysates show detectible SEC14L3 expression by Western blot

  • Human testis, breast cancer, and gastric cancer tissues show SEC14L3 immunoreactivity by IHC

When selecting a model system, researchers should consider the expression profile of SEC14L3 and related family members in their system of interest, as expression patterns may vary across species and tissues.