SEC14L4 Antibody

What is SEC14L4 and why is it important to study?

SEC14L4 (SEC14-like lipid binding 4) is a probable hydrophobic ligand-binding protein involved in the transport of hydrophobic ligands like tocopherol, squalene, and phospholipids. In humans, the canonical protein has 406 amino acid residues and a molecular mass of 46.6 kDa, with up to two different isoforms reported .

This protein belongs to the SEC14-like family, representing mammalian Golgi dynamics proteins uniquely found in all eukaryotic genomes . Its importance in research stems from its role in lipid homeostasis and cellular lipid transport, with dysregulation linked to various conditions including metabolic disorders, neurodegenerative diseases, and cancer . Recent studies have specifically implicated high SEC14L4 expression as a prognostic indicator of poor outcomes in esophageal squamous cell cancer (ESCC) .

What types of SEC14L4 antibodies are available for research purposes?

Multiple types of SEC14L4 antibodies are available for research applications:

Most available antibodies target human SEC14L4, though some cross-react with other species. Region-specific antibodies, such as those targeting the N-terminal region, are also available for specialized applications .

What are the most common applications for SEC14L4 antibodies?

Based on commercially available options, SEC14L4 antibodies are most commonly used in the following applications:

• Western Blot (WB): The most frequent application, used to detect and quantify SEC14L4 protein expression in cell or tissue lysates.

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of SEC14L4.

• Immunohistochemistry (IHC): Both paraffin-embedded (IHC-p) and frozen sections (IHC-fr) for localizing SEC14L4 in tissue specimens.

• Immunocytochemistry (ICC)/Immunofluorescence (IF): For visualizing SEC14L4 distribution within cells.

• Flow Cytometry (FCM): Less common but used for detecting SEC14L4 in cell populations.

• Immunoprecipitation (IP): For isolating SEC14L4 protein complexes .

What are the optimal storage conditions for SEC14L4 antibodies?

Most SEC14L4 antibodies are supplied in liquid form with a storage buffer typically consisting of 50% glycerol, 0.01M PBS, pH 7.4, and a preservative such as 0.03% Proclin 300 . For optimal stability and activity, these antibodies should be:

• Stored at -20°C for long-term storage

• Avoided repeated freeze-thaw cycles (aliquoting upon receipt is recommended)

• Kept at 4°C for short-term use (1-2 weeks)

• Protected from light, especially if conjugated to fluorophores

How does SEC14L4 expression correlate with disease states, particularly in cancer?

Recent research has revealed significant associations between SEC14L4 expression and cancer progression:

High expression of SEC14L4 has been identified as a prognostic indicator of poor outcomes in patients with esophageal squamous cell cancer (ESCC) . The biological mechanism appears related to SEC14L4's role in lipid metabolism and transport, as alterations in lipid metabolism and glycerophospholipids in cancer cells can affect the immune system response .

This correlation makes SEC14L4 a potential therapeutic target and biomarker in ESCC and possibly other cancers. Researchers investigating SEC14L4 in cancer contexts should consider:

• Comparing expression levels between tumor and adjacent normal tissues

• Correlating expression with clinical parameters like tumor stage, grade, and patient survival

• Investigating the functional consequences of SEC14L4 knockdown or overexpression in cancer cell lines

• Exploring the relationship between SEC14L4 and specific lipid profiles in the tumor microenvironment

What are the key methodological considerations when validating SEC14L4 antibody specificity?

Ensuring antibody specificity is critical for reliable research outcomes. For SEC14L4 antibodies, consider these validation approaches:

• Positive and negative controls:

  • Positive: Use tissues/cells known to express SEC14L4 (e.g., kidney and liver tissues have shown positive immunohistochemical staining)

  • Negative: Use SEC14L4 knockout models or siRNA-treated cells

• Isoform specificity testing:

  • Since up to 2 different isoforms have been reported for SEC14L4 , determine if your antibody recognizes specific or all isoforms

  • Consider western blot analysis to confirm detection of the expected 46.6 kDa band

• Cross-reactivity assessment:

  • Test reactivity with other SEC14 family members (SEC14L2/L3/L4), as some antibodies may cross-react

  • Perform immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody

• Technical validation across methods:

  • Compare antibody performance across multiple techniques (e.g., if positive in WB, confirm with IHC)

  • Use multiple antibodies targeting different epitopes of SEC14L4 to confirm findings

How can researchers distinguish between SEC14L4 and other SEC14-like family members?

The SEC14-like family contains several members with structural and functional similarities. To distinguish SEC14L4 specifically:

• Epitope selection:

  • Choose antibodies targeting regions with minimal sequence homology to other SEC14 family proteins

  • N-terminal region antibodies are available and may offer better specificity

• Molecular weight confirmation:

  • SEC14L4: 46.6 kDa

  • Validate molecular weight precisely using SDS-PAGE with appropriate markers

• Expression pattern analysis:

  • Different SEC14 family members have distinct tissue expression patterns

  • SEC14L4 gene orthologs have been reported in mouse and chimpanzee species

• Functional assays:

  • Develop assays specific to SEC14L4's role in lipid transport

  • Use specific lipid binding assays to differentiate function from other family members

What are the emerging techniques for studying SEC14L4's role in lipid metabolism?

As a protein involved in lipid transport and metabolism, SEC14L4 research benefits from specialized techniques:

• Lipidomics approaches:

  • Mass spectrometry-based lipidomics to identify specific lipids transported by SEC14L4

  • Lipid binding assays to characterize affinity for tocopherol, squalene, and phospholipids

• Live-cell imaging:

  • Fluorescently tagged SEC14L4 to track intracellular movement

  • FRET-based assays to monitor lipid transfer activities

• Structural biology:

  • Crystallography or cryo-EM to determine SEC14L4 structure, especially the lipid-binding pocket

  • Computational modeling of ligand interactions

• Genome editing:

  • CRISPR-Cas9 to generate SEC14L4 knockout or knock-in models

  • Site-directed mutagenesis to identify key residues for lipid binding

What are the optimal conditions for Western blot detection of SEC14L4?

For successful Western blot detection of SEC14L4 (46.6 kDa), consider these protocol elements:

• Sample preparation:

  • Extract proteins using lysis buffers containing protease inhibitors

  • For membrane-associated proteins like SEC14L4, include detergents like NP-40 or Triton X-100

• Gel electrophoresis:

  • Use 10-12% polyacrylamide gels for optimal separation around 46.6 kDa

  • Load appropriate positive controls (e.g., liver or kidney tissue lysates)

• Transfer and blocking:

  • PVDF membranes typically work well for hydrophobic proteins

  • Block with 5% non-fat milk or BSA in TBST

• Antibody incubation:

  • Primary antibody dilution: typically 1:500-1:2000 (optimize for specific antibody)

  • Incubate overnight at 4°C for best results

  • Secondary antibody: 1:5000-1:10000, species-matched to primary antibody host

• Detection:

  • Both chemiluminescence and fluorescence detection systems are suitable

  • For quantitative analysis, consider fluorescence-based detection

How should researchers optimize immunohistochemistry protocols for SEC14L4 detection?

Based on successful IHC applications reported for SEC14L4 antibodies:

• Tissue preparation:

  • Both paraffin-embedded and frozen sections have been used successfully

  • Paraffin sections typically require antigen retrieval

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is commonly effective

  • Test both citrate and EDTA-based retrieval solutions

• Antibody dilution and incubation:

  • Starting dilution range: 1:20-1:200 as recommended

  • Optimize using positive control tissues (kidney and liver tissues have shown positive staining)

• Detection system:

  • HRP/DAB systems work well for brightfield microscopy

  • For fluorescence, select secondary antibodies with appropriate fluorophores

• Controls:

  • Include positive tissue controls (human kidney and liver)

  • Include negative controls (omitting primary antibody)

  • Consider peptide competition assays for specificity verification

How can researchers quantitatively assess SEC14L4 expression levels in experimental samples?

For rigorous quantitative analysis of SEC14L4 expression:

• Protein quantification methods:

  • Western blot with densitometry analysis (normalize to loading controls)

  • ELISA (for absolute quantification, use standard curves with recombinant SEC14L4)

  • Quantitative immunofluorescence with appropriate controls

• mRNA quantification:

  • RT-qPCR (design primers specific to SEC14L4, avoiding other family members)

  • RNAseq for broader expression context

  • In situ hybridization for spatial expression analysis

• Digital pathology approaches:

  • For IHC analysis, use digital image analysis software to quantify DAB staining intensity

  • H-score or Allred scoring systems for semi-quantitative assessment

  • Multiplex immunofluorescence for co-expression with other proteins

• Statistical considerations:

  • Use appropriate statistical tests based on data distribution

  • Include sufficient biological replicates (minimum n=3)

  • Consider power analysis to determine appropriate sample sizes

Why might Western blot detection of SEC14L4 show multiple bands?

Multiple bands when probing for SEC14L4 can occur for several reasons:

• Isoform detection: Up to 2 different isoforms have been reported for SEC14L4 .

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can alter apparent molecular weight.

• Proteolytic degradation: Incomplete protease inhibition during sample preparation.

• Cross-reactivity: Some antibodies may detect other SEC14 family members (SEC14L2/L3) .

• Non-specific binding: Particularly with polyclonal antibodies.

Troubleshooting approaches:

• Optimize protein extraction with fresh, complete protease inhibitors

• Test different blocking reagents (milk vs. BSA)

• Increase washing stringency and duration

• Try antibodies targeting different epitopes

• Use gradient gels for better separation

What strategies can overcome weak or absent signal in immunohistochemistry?

If experiencing weak or no signal when detecting SEC14L4 in tissues:

• Antigen retrieval optimization:

  • Test multiple retrieval methods (heat vs. enzymatic)

  • Adjust retrieval time and temperature

  • Try different buffer systems (citrate, EDTA, Tris)

• Antibody concentration:

  • Titrate antibody concentrations (starting with manufacturer recommendations of 1:20-1:200)

  • Consider longer incubation times (overnight at 4°C)

• Detection system enhancement:

  • Use polymer-based detection systems for signal amplification

  • Consider tyramide signal amplification for very low abundance targets

  • Try biotin-streptavidin systems if not already used

• Sample quality assessment:

  • Verify tissue fixation quality with other antibodies

  • Check positive control tissues (kidney and liver have shown positive staining)

  • Consider tissue age and storage conditions

How can researchers differentiate between specific and non-specific staining in immunohistochemistry?

Distinguishing true SEC14L4 signal from background:

• Control experiments:

  • Peptide competition/neutralization assays

  • SEC14L4 knockdown/knockout tissues as negative controls

  • Comparison with mRNA expression patterns (ISH or public databases)

• Pattern analysis:

  • SEC14L4 is suspected to be involved in lipid transport, suggesting intracellular/membrane localization

  • Compare staining pattern with published data and expected cellular locations

• Antibody validation:

  • Use multiple antibodies targeting different epitopes

  • Compare staining patterns across different antibodies

  • Validate findings with orthogonal methods (WB, IF)

• Technical controls:

  • No primary antibody control

  • Isotype control antibody

  • Gradient of antibody concentrations to identify optimal signal-to-noise ratio

How should researchers design experiments to investigate SEC14L4's role in cancer progression?

Based on SEC14L4's association with poor outcomes in ESCC , researchers might consider:

• Expression correlation studies:

  • Compare SEC14L4 expression between tumor and adjacent normal tissues

  • Correlate expression with clinical parameters (stage, grade, metastasis)

  • Analyze public databases (TCGA, GEO) for broader context across cancer types

• Functional studies:

  • Knockdown/overexpression models to assess effects on:

    • Proliferation

    • Migration/invasion

    • Resistance to therapy

    • Lipid metabolism alterations

• Mechanistic investigations:

  • Identify SEC14L4 interaction partners via co-IP/mass spectrometry

  • Map affected signaling pathways

  • Characterize lipid profile changes using lipidomics

• In vivo models:

  • Generate SEC14L4 knockout or overexpression mouse models

  • Assess tumor development, progression, and metastasis

  • Evaluate response to standard therapies

What is the emerging evidence for SEC14L4's role in the immune response within disease contexts?

The search results indicate that alterations in lipid metabolism and glycerophospholipids in cancer cells can affect the immune system response . For SEC14L4 specifically:

• Immune contexture analysis:

  • Correlate SEC14L4 expression with immune cell infiltration

  • Assess relationship with specific immune cell populations (T cells, macrophages)

  • Evaluate association with immune checkpoint markers

• Lipid mediator investigation:

  • Analyze how SEC14L4-mediated lipid transport affects immunomodulatory lipids

  • Examine eicosanoid production and signaling

  • Assess impact on membrane composition of immune cells

• Therapeutic implications:

  • Evaluate whether SEC14L4 inhibition sensitizes to immunotherapy

  • Assess combination approaches targeting both SEC14L4 and immune checkpoints

  • Consider SEC14L4 as a biomarker for immunotherapy response prediction

How can researchers effectively design studies to elucidate SEC14L4's role in lipid metabolism disorders?

As SEC14L4 is involved in lipid transport, researchers investigating metabolic disorders should consider:

• Expression profiling:

  • Analyze SEC14L4 expression in tissues relevant to lipid metabolism (liver, adipose)

  • Compare expression levels in normal vs. metabolic disease states

  • Assess correlation with specific lipid profile abnormalities

• Metabolic phenotyping:

  • Generate SEC14L4 transgenic models

  • Characterize effects on:

    • Serum lipid profiles

    • Glucose homeostasis

    • Liver fat accumulation

    • Response to high-fat diet challenges

• Cellular metabolism studies:

  • Assess impact on lipid droplet formation and dynamics

  • Characterize effects on fatty acid uptake, synthesis and oxidation

  • Measure membrane lipid composition changes

• Therapeutic targeting potential:

  • Screen for small molecule modulators of SEC14L4

  • Evaluate effects of targeting SEC14L4 in metabolic disease models

  • Assess pharmacological vs. genetic inhibition outcomes

What novel technical approaches might advance our understanding of SEC14L4 function?

Emerging technologies could provide new insights into SEC14L4 biology:

• Single-cell techniques:

  • scRNA-seq to identify cell populations with high SEC14L4 expression

  • Spatial transcriptomics to map SEC14L4 expression in tissue context

  • CyTOF or single-cell proteomics for protein-level analysis

• Advanced imaging:

  • Super-resolution microscopy to visualize SEC14L4 subcellular localization

  • Correlative light and electron microscopy (CLEM) for ultrastructural context

  • Live-cell imaging with lipid probes to track SEC14L4-mediated transport

• Structural biology innovations:

  • AlphaFold or other AI prediction tools to model SEC14L4 structure

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic protein regions

  • Small-angle X-ray scattering for solution structure analysis

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, lipidomics)

  • Network analysis to place SEC14L4 in broader cellular pathways

  • Computational modeling of lipid trafficking processes

What are the key unresolved questions regarding SEC14L4 biology?

Despite current knowledge, several fundamental questions remain:

• Substrate specificity:

  • Which specific lipid species does SEC14L4 preferentially transport?

  • How does substrate binding affect protein conformation?

  • What determines specificity among SEC14 family members?

• Regulatory mechanisms:

  • How is SEC14L4 expression and activity regulated?

  • What post-translational modifications affect function?

  • What are the key transcription factors controlling expression?

• Physiological roles:

  • What is the normal physiological function in different tissues?

  • How do the two reported isoforms differ functionally?

  • What phenotypes result from SEC14L4 deficiency?

• Disease relevance beyond cancer:

  • Are SEC14L4 alterations involved in neurodegenerative diseases?

  • What is its role in metabolic syndrome and liver diseases?

  • Could SEC14L4 be implicated in inflammatory disorders?

By addressing these questions, researchers can significantly advance our understanding of SEC14L4 biology and its potential as a therapeutic target.