SEC14L2 Antibody, HRP conjugated

What is SEC14L2 and why is it significant in research?

SEC14L2 functions as a carrier protein that binds to hydrophobic molecules and promotes their transfer between different cellular sites. It exhibits high affinity binding to α-tocopherol and weaker affinity to other tocopherols and tocotrienols. The protein may possess transcriptional activatory activity via its association with α-tocopherol and potentially regulates cholesterol biosynthesis by increasing the transfer of squalene to metabolically active pools in cells. SEC14L2 has garnered significant research interest due to its role in enabling pan-genotype HCV replication in cell culture and its associations with various neoplasms and liver diseases .

What detection methods are most effective for SEC14L2 using HRP-conjugated antibodies?

HRP-conjugated SEC14L2 antibodies are particularly effective in Western blotting, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) applications. For Western blotting, optimal dilutions typically range from 1:500 to 1:5000, while IHC applications generally require 1:200 to 1:500 dilutions. The detection is commonly achieved through a secondary antibody system where the primary anti-SEC14L2 antibody is detected by a biotinylated secondary antibody and visualized using an HRP-conjugated SP system. This methodology provides high sensitivity and specificity when properly optimized for each experimental context .

How do monoclonal and polyclonal SEC14L2 antibodies differ in research applications?

Monoclonal SEC14L2 antibodies (such as H-4) offer high specificity by recognizing a single epitope, making them ideal for applications requiring consistent lot-to-lot reproducibility and minimal background. They are particularly valuable in detecting specific SEC14L2 isoforms. In contrast, polyclonal antibodies recognize multiple epitopes, potentially offering greater sensitivity by binding to various regions of the target protein. For HRP-conjugated applications, monoclonal antibodies typically provide cleaner Western blot results with less background, while polyclonal antibodies may offer advantages in IHC applications where signal amplification is beneficial. The choice depends on experimental requirements, with monoclonals preferred for isoform-specific detection and polyclonals for applications where protein conformation may be altered .

What are the optimal protocols for using HRP-conjugated SEC14L2 antibodies in Western blotting?

For Western blot applications using HRP-conjugated SEC14L2 antibodies, optimal results are achieved with careful protocol optimization. Begin with sample preparation using whole cell lysates (such as PC3 cells, which show high expression). Use 4.28μg/ml as a starting antibody concentration, with standard SDS-PAGE separation followed by transfer to PVDF or nitrocellulose membranes. For primary incubation, dilute antibodies in the range of 1:500-1:5000 in blocking buffer containing 1% BSA, and incubate overnight at 4°C. For non-directly conjugated antibodies, follow with a goat polyclonal to rabbit IgG at approximately 1/50000 dilution. The expected band size for SEC14L2 is 47 kDa, though 45 kDa and 37 kDa bands may also be observed, potentially representing different isoforms. Signal development using enhanced chemiluminescence (ECL) substrates optimized for HRP should yield clear bands with minimal background when proper blocking and washing steps are employed .

How should SEC14L2 antibodies be optimized for immunohistochemistry applications?

For immunohistochemistry applications, SEC14L2 antibodies require specific optimization protocols. After dewaxing and hydrating paraffin-embedded tissues, antigen retrieval is critical and most effectively performed via high pressure in citrate buffer (pH 6.0). Section blocking with 10% normal goat serum for 30 minutes at room temperature minimizes non-specific binding. Dilute the primary SEC14L2 antibody at approximately 1:400 in 1% BSA and incubate at 4°C overnight for optimal epitope binding. For visualization, employ a biotinylated secondary antibody followed by an HRP-conjugated SP system. This methodology has been successfully applied to various tissue types, including prostate tissue and colon cancer samples, with clear cellular localization of SEC14L2. Signal specificity should be validated using appropriate positive and negative controls, particularly tissues with known differential expression of SEC14L2 (liver, brain, intestine, and prostate show high expression levels) .

What considerations are important when designing immunofluorescence experiments with SEC14L2 antibodies?

For immunofluorescence applications using SEC14L2 antibodies, cell fixation methodology significantly impacts results. Optimal protocols include fixation with 4% formaldehyde followed by permeabilization using 0.2% Triton X-100. Block cells with 10% normal goat serum to reduce background. The SEC14L2 antibody should be diluted approximately 1:133 (though ranges from 1:50-1:200 may be optimal depending on the specific antibody) and incubated overnight at 4°C. For visualization, Alexa Fluor 488-conjugated secondary antibodies provide excellent signal-to-noise ratios. When interpreting results, it's critical to recognize that SEC14L2 predominantly localizes to the cytoplasm under normal conditions, while α-tocopherol exposure induces nuclear translocation. This subcellular redistribution phenomenon must be considered when analyzing images, particularly in experiments involving vitamin E treatment or oxidative stress conditions .

How can SEC14L2 antibodies be utilized in HCV research?

SEC14L2 antibodies serve as crucial tools in HCV research due to the protein's role in enabling pan-genotype HCV replication in cell culture. When establishing experimental models, researchers should consider both endogenous SEC14L2 detection and validation of transgene expression in engineered cell lines. For studying HCV replication mechanisms, Western blotting with HRP-conjugated SEC14L2 antibodies can confirm protein expression levels in Huh-7.5, Huh-7, or Hep3B/miR122 cells transfected with SEC14L2 expression vectors. Immunofluorescence applications are particularly valuable for co-localization studies examining the relationship between SEC14L2 and viral replication complexes. When designing experiments, it's essential to note that SEC14L2's effect on HCV replication is dose-dependent and requires continuous expression, necessitating careful monitoring of protein levels throughout experimental timeframes. For mechanistic studies, SEC14L2 antibodies can help elucidate how the protein enhances vitamin E-mediated protection against lipid peroxidation, which appears to be the primary mechanism by which it promotes HCV infection .

What is the relationship between SEC14L2 and lipid peroxidation, and how can antibodies help investigate this?

SEC14L2 plays a significant role in protecting against lipid peroxidation, a relationship that can be effectively investigated using SEC14L2 antibodies in combination with lipid peroxidation assays. Research protocols should include quantification of malondialdehyde levels (a secondary product of lipid peroxidation) in SEC14L2-expressing versus control cells, alongside Western blot confirmation of SEC14L2 expression levels. The protein has been shown to reduce the inhibitory effect of lipophilic oxidants on processes like HCV replication, but does not affect inhibition by interferon-alpha or cyclosporine A, indicating selective protection against lipid peroxidation. When designing experiments to investigate this relationship, researchers should consider SEC14L2's high affinity for α-tocopherol (vitamin E), as this interaction appears central to its antioxidant function. Immunoprecipitation with SEC14L2 antibodies followed by analysis of co-precipitated lipids can provide insights into the direct binding partners involved in this protective mechanism. Additionally, researchers should note that SEC14L2 expression impacts the effectiveness of certain antiviral compounds, particularly NS3 protease and NS5B polymerase inhibitors, by altering the cellular redox environment .

How can different SEC14L2 domains be analyzed using domain-specific antibodies?

Analysis of SEC14L2's functional domains requires careful selection of domain-specific antibodies or epitope-mapped antibodies that recognize distinct regions. SEC14L2 consists of three key domains: an N-terminal CRAL-TRIO domain (aa 12-60) that binds small lipophilic molecules, a SEC14-like domain (aa 76-245) containing a hydrophobic ligand-binding pocket, and a C-terminal GOLD domain (aa 300-380) involved in protein-protein interactions. When designing domain-function studies, researchers should verify whether their antibodies recognize specific domains or conformational epitopes spanning multiple regions. For studies involving mutant constructs, Western blotting with multiple antibodies recognizing different epitopes can confirm expression of truncated proteins. Immunoprecipitation experiments can determine which domains are necessary for interactions with partner proteins. It's important to note that research has shown that multiple SEC14L2-specific determinants are required for its function in processes like HCV replication, as neither deletion mutants nor SEC14L2/L4 chimeras conferred HCV permissiveness to cells, suggesting complex structural requirements for full functionality .

What are common issues with non-specific binding when using HRP-conjugated SEC14L2 antibodies, and how can they be addressed?

Non-specific binding with HRP-conjugated SEC14L2 antibodies typically manifests as multiple unexpected bands in Western blots or diffuse background staining in IHC/IF applications. To address these issues, implement a systematic optimization approach:

• For Western blotting:

  • Extend blocking time to 2 hours using 5% BSA or milk in TBST

  • Increase wash duration and frequency (5 × 10 minutes with TBST)

  • Titrate antibody concentration (starting with higher dilutions of 1:5000)

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer

  • Consider using gradient gels to better resolve SEC14L2 isoforms (47, 45, and 37 kDa)

• For IHC/IF applications:

  • Perform more stringent antigen retrieval optimization

  • Use higher dilutions of primary antibody (1:500-1:1000)

  • Extend blocking with 10% normal serum from the same species as secondary antibody

  • Include 0.1% Triton X-100 and 0.3% BSA in antibody diluent

  • Validate specificity using SEC14L2 knockout or knockdown controls

The cross-reactivity with SEC14L3 and SEC14L4 should be considered when evaluating antibody specificity, particularly in tissues where these related proteins may be expressed .

How can researchers distinguish between SEC14L2 and related family members (SEC14L3, SEC14L4) in their experiments?

Distinguishing between SEC14L2 and its family members requires careful antibody selection and experimental design. First, perform in silico analysis of antibody epitopes against sequence alignments of SEC14L2, SEC14L3, and SEC14L4 to identify regions of high homology that might lead to cross-reactivity. When designing experiments, include positive controls expressing each family member individually to establish antibody specificity profiles. Western blotting can sometimes resolve family members based on molecular weight differences, though subtle variations may require high-resolution gradient gels. For tissue expression studies, it's important to note that while SEC14L2 is predominantly expressed in liver, brain, intestine, and prostate, the expression patterns of SEC14L3 and SEC14L4 differ. For functional studies, consider that while SEC14L3 allowed low-level HCV replication, SEC14L4 had no effect in this system, providing a functional readout to distinguish these proteins. RNA-based approaches (qPCR with isoform-specific primers) can complement protein detection methods to verify which family member is actually expressed in a given experimental system .

What are the critical factors affecting reproducibility when using SEC14L2 antibodies in different applications?

Reproducibility challenges with SEC14L2 antibodies stem from several critical factors that require systematic control:

• Antibody lot-to-lot variability:

  • Characterize each new lot against a reference standard

  • Maintain consistent sourcing when possible

  • Document lot numbers used for all experiments

• Sample preparation inconsistencies:

  • Standardize cell lysis buffers and conditions

  • For tissue samples, control fixation time precisely (10% neutral buffered formalin for 24-48 hours)

  • Maintain consistent protein loading (20-30μg for Western blots)

• Protocol variability:

  • Document detailed protocols including blocking agent percentages, incubation times and temperatures

  • For IHC, standardize antigen retrieval methods (citrate buffer pH 6.0 under high pressure)

  • Control incubation temperature fluctuations (<±1°C)

• SEC14L2 post-translational modifications:

  • Consider that phosphorylation by protein kinase A and protein kinase C modifies SEC14L2 function

  • These modifications may affect antibody recognition depending on epitope location

  • Where relevant, use phospho-specific antibodies or phosphatase treatments

• Expression level variations:

  • SEC14L2 expression is influenced by α-tocopherol and oxidative stress conditions

  • Standardize culture conditions, particularly vitamin E content in media

  • Document passage number of cell lines as expression patterns may drift

Maintaining these controls across experiments significantly improves reproducibility when working with SEC14L2 antibodies .

What methodological approaches can be used to validate SEC14L2 antibody specificity for critical research applications?

Validating SEC14L2 antibody specificity requires a multi-faceted approach to ensure reliable research outcomes:

• Genetic validation approaches:

  • CRISPR/Cas9 knockout cells as negative controls

  • siRNA/shRNA knockdown with titrated reduction in signal

  • Heterologous expression systems with controlled SEC14L2 overexpression

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide

  • Observe dose-dependent signal reduction

  • Include non-specific peptide controls

• Orthogonal detection methods:

  • Compare results using antibodies targeting different SEC14L2 epitopes

  • Correlate protein detection with mRNA levels by RT-qPCR

  • Mass spectrometry validation of immunoprecipitated proteins

• Cross-reactivity assessment:

  • Test against recombinant SEC14L3 and SEC14L4

  • Evaluate species cross-reactivity if working with murine models

  • Note that murine SEC14L2 was permissive to wild-type HCV, suggesting functional conservation

• Application-specific validation:

  • For Western blots: confirm expected molecular weight (47 kDa)

  • For IHC: validate tissue expression patterns (high in liver, brain, intestine, prostate)

  • For IP: confirm pulled-down proteins by mass spectrometry

Implementing these validation strategies provides crucial evidence of antibody specificity, particularly important for publication-quality research involving SEC14L2 .

How can researchers quantitatively assess SEC14L2 expression levels in different experimental conditions?

Quantitative assessment of SEC14L2 expression requires rigorous methodological approaches tailored to the experimental context:

Western Blot Quantification:

qPCR Correlation:
For transcriptional studies, correlate protein expression with mRNA levels using qPCR. Primer sequences targeting SEC14L2 should span exon-exon junctions to avoid genomic DNA amplification. Reference genes should be validated for stability in the experimental conditions.

Flow Cytometry:
For single-cell resolution, intracellular staining for SEC14L2 can be performed after fixation and permeabilization. This approach allows quantification of expression heterogeneity within populations and can be combined with surface markers for cell-type specific analysis.

Image-Based Quantification:
For microscopy-based quantification of immunofluorescence, use consistent exposure settings and automated image analysis workflows. Measure integrated density or mean fluorescence intensity within defined cellular regions (total cell, cytoplasm, nucleus). Automated segmentation based on nuclear and cellular markers improves reproducibility.

These approaches should be validated using dose-response controls, such as doxycycline-inducible SEC14L2 expression systems, which have demonstrated a clear dose-dependent relationship between SEC14L2 levels and functional outcomes like HCV replication .

How can SEC14L2 antibodies contribute to understanding the protein's role in disease mechanisms?

SEC14L2 antibodies provide critical tools for investigating the protein's involvement in various disease mechanisms, particularly in neoplasms and viral infections. In cancer research, immunohistochemistry with SEC14L2 antibodies can reveal expression patterns across tumor types and stages, with documented associations to prostatic diseases, breast neoplasms, and hepatocellular carcinoma. Researchers should employ tissue microarrays to systematically compare expression across multiple patient samples with appropriate controls. For mechanistic studies, co-immunoprecipitation experiments using SEC14L2 antibodies can identify disease-relevant protein interaction partners that may be altered in pathological states. In HCV research, SEC14L2 antibodies are instrumental in confirming expression levels when studying the protein's role in enabling viral replication. The antioxidant function of SEC14L2 appears particularly relevant to disease mechanisms, as it enhances vitamin E-mediated protection against lipid peroxidation. This protective effect influences the efficacy of certain antiviral compounds and may have broader implications for diseases involving oxidative stress. Carefully designed immunofluorescence studies can reveal whether SEC14L2's subcellular localization is altered in disease states, potentially providing insights into functional changes .

What experimental design considerations are important when studying SEC14L2 in viral infection models?

When designing experiments to study SEC14L2 in viral infection models, particularly HCV, several critical considerations must be addressed:

• Cell line selection:

  • Huh-7.5 cells show highest permissiveness when expressing SEC14L2

  • Huh-7 and Hep3B/miR122 cells are also suitable but with lower efficiency

  • Primary hepatocytes should be evaluated for endogenous SEC14L2 expression

• Expression system design:

  • Use doxycycline-inducible systems for controlled expression

  • Continuous expression is required for maintaining viral replication

  • Titrate expression levels to establish dose-dependent relationships

• Viral strain considerations:

  • SEC14L2 enables replication of all HCV genotypes (1a, 1b, 2a, 3a, 4a, 5a)

  • Patient sera can be used directly in SEC14L2-expressing cells

  • Compare non-adapted clinical isolates with laboratory-adapted strains

• Analytical approaches:

  • Viral replication monitoring via qRT-PCR, luciferase reporters, or colony formation assays

  • SEC14L2 expression confirmation via Western blotting with HRP-conjugated antibodies

  • Colocalization studies via immunofluorescence for SEC14L2 and viral proteins

• Mechanistic investigations:

  • Include lipid peroxidation measurements (malondialdehyde quantification)

  • Test protection against lipophilic oxidants specifically

  • Evaluate α-tocopherol levels and supplementation effects

• Controls:

  • SEC14L3 and SEC14L4 expression constructs as functional comparisons

  • SEC14L2 deletion mutants to identify essential domains

  • Anti-CD81 antibody to block viral entry as specificity control

These design elements enable robust investigation of SEC14L2's role in facilitating viral infection while providing appropriate controls for specificity and mechanistic insights .

What are the comparative advantages of using directly HRP-conjugated SEC14L2 antibodies versus detection with secondary antibodies?

When comparing directly HRP-conjugated SEC14L2 antibodies to traditional primary-secondary antibody detection systems, researchers should consider several key advantages and limitations:

Directly HRP-conjugated SEC14L2 antibodies:

• Advantages: Simplified workflow with fewer incubation steps and reduced total protocol time (saves approximately 2-4 hours); minimized species cross-reactivity issues in co-staining experiments; reduced background signal from non-specific secondary antibody binding; more consistent results with less variability introduced by secondary antibody inconsistencies; enhanced sensitivity for low-abundance targets due to direct conjugation.

• Limitations: Reduced signal amplification compared to secondary systems; limited flexibility for signal strength adjustments; potential reduction in antibody binding efficiency due to HRP modification; higher cost per experiment; typically shorter shelf-life than unconjugated antibodies.

Primary SEC14L2 antibody with secondary HRP detection:

• Advantages: Signal amplification through multiple secondary antibodies binding each primary antibody (3-10× signal enhancement); flexibility to adjust signal by modifying secondary antibody concentration; ability to use the same primary antibody with different detection systems (HRP, fluorescence, etc.); cost-effectiveness for multiple experiments; typically better shelf stability.

• Limitations: Longer protocols with additional incubation and wash steps; increased background potential from non-specific secondary antibody binding; potential cross-reactivity issues in multi-labeling experiments; more variables to optimize.

For SEC14L2 detection specifically, directly conjugated antibodies excel in multi-labeling studies examining co-localization with other proteins, while the traditional approach may be preferable for detecting low-expression levels in tissue samples .

How can phosphorylation status of SEC14L2 be effectively analyzed in experimental systems?

Analyzing SEC14L2 phosphorylation status requires specific methodological approaches that address both detection and functional implications:

• Phosphorylation-specific antibodies:

  • Use antibodies specifically recognizing phosphorylated forms of SEC14L2

  • Validate specificity using lambda phosphatase-treated controls

  • Consider developing custom phospho-specific antibodies against known PKA and PKC target sites

• Phosphorylation detection methods:

  • Phos-tag SDS-PAGE gels to separate phosphorylated from non-phosphorylated forms

  • 2D gel electrophoresis (IEF followed by SDS-PAGE) to resolve phospho-isoforms

  • Mass spectrometry-based phosphopeptide mapping for site identification

• Functional kinase assays:

  • In vitro kinase assays with purified SEC14L2 and PKA or PKC

  • Cell-based assays with kinase activators (forskolin for PKA, PMA for PKC)

  • Inhibitor studies using selective PKA inhibitors (H-89) or PKC inhibitors (Gö6983)

• Phosphomimetic mutants:

  • Generate serine/threonine to aspartate or glutamate mutations

  • Create phospho-null mutations (serine/threonine to alanine)

  • Compare functional outcomes in relevant assays (HCV replication, α-tocopherol binding)

• Physiological context:

  • Analyze phosphorylation changes in response to oxidative stress

  • Examine correlation between phosphorylation and subcellular localization

  • Investigate phosphorylation dynamics during viral infection

Since SEC14L2 activity is known to be modulated by phosphorylation through PKA and PKC pathways, these approaches provide critical insights into regulatory mechanisms controlling its various functions in lipid metabolism and antioxidant protection .

What methodological approaches can be used to study SEC14L2 interactions with α-tocopherol and other binding partners?

Studying SEC14L2 interactions with α-tocopherol and other partners requires specialized biochemical and cellular approaches:

• Direct binding assays:

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of interaction

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Fluorescence quenching or anisotropy with labeled ligands

  • Radiolabeled ligand binding assays for high sensitivity

• Pull-down assays using SEC14L2 antibodies:

  • Immunoprecipitation followed by mass spectrometry for protein partners

  • Co-IP with Western blotting for specific suspected interactions

  • For lipid interactions, extract and analyze bound lipids by LC-MS/MS

  • Crosslinking approaches for transient interactions

• Structural methods:

  • X-ray crystallography of SEC14L2 with bound ligands

  • NMR spectroscopy for dynamic interaction mapping

  • Hydrogen-deuterium exchange mass spectrometry for binding interfaces

• Cellular approaches:

  • FRET-based approaches for protein-protein interactions

  • Fluorescently-labeled α-tocopherol analogs for tracking

  • Subcellular fractionation with quantification of SEC14L2 and binding partners

  • Proximity ligation assays for in situ interaction detection

• Functional competition studies:

  • Competitive binding assays between α-tocopherol and other tocopherols/tocotrienols

  • Dose-dependent functional readouts (HCV replication, antioxidant protection)

  • Mutagenesis of binding pocket residues with functional analysis

These methodologies provide complementary approaches to characterize SEC14L2's interactions with both small molecules and protein partners, yielding insights into the molecular mechanisms underlying its diverse cellular functions. Particular attention should be paid to the SEC14-like domain (aa 76-245) containing the hydrophobic ligand-binding pocket, which is critical for α-tocopherol binding .