SEC14L2 Antibody, FITC conjugated

What is SEC14L2 and why is it significant in biological research?

SEC14L2 (SEC14-like protein 2), also known as alpha-tocopherol-associated protein (TAP), supernatant protein factor (SPF), or squalene transfer protein, is a 36.6 kDa carrier protein that binds to hydrophobic molecules and promotes their transfer between different cellular sites. It binds with high affinity to alpha-tocopherol and with weaker affinity to other tocopherols and tocotrienols. SEC14L2 may have transcriptional activatory activity via its association with alpha-tocopherol and plays a role in regulating cholesterol biosynthesis by binding squalene structures . Recent research has identified SEC14L2 as a GTPase protein that transduces Wnt signals from Frizzled to phospholipase C (PLC), playing a critical role in the non-canonical Wnt/Ca²⁺ signaling pathway important for embryonic development and tissue formation .

What are the key specifications of commercially available SEC14L2 antibodies, including FITC-conjugated variants?

Most commercial SEC14L2 antibodies are rabbit polyclonal antibodies raised against human SEC14L2. FITC-conjugated variants like MBS7109006 are part of matching pairs with unconjugated antibodies (MBS7109004) . These antibodies typically:

• Have molecular reactivity to human SEC14L2 (some with cross-reactivity to mouse)

• Are purified using protein G or antigen affinity methods (>95% purity)

• Have applications in Western Blot (1:500-1:5000 dilution), Immunohistochemistry (1:200-1:500), Immunofluorescence (1:50-1:200), and ELISA

• Are stored in buffers containing glycerol and preservatives

• Should be stored at -20°C to maintain stability

What is the difference between various conjugates of SEC14L2 antibodies and when should I choose FITC-conjugated variants?

FITC-conjugated SEC14L2 antibodies should be chosen when direct visualization without secondary antibodies is needed, particularly for immunofluorescence studies, flow cytometry, or when multiplexing with other fluorophores that have distinct spectral characteristics. The direct conjugation eliminates cross-reactivity issues with secondary antibodies and reduces the number of incubation steps in protocols .

What are optimal sample preparation methods for SEC14L2 immunofluorescence studies using FITC-conjugated antibodies?

For effective immunofluorescence with FITC-conjugated SEC14L2 antibodies:

• Cell Fixation: Fix cells in 4% formaldehyde for 10-15 minutes at room temperature.

• Permeabilization: Use 0.2% Triton X-100 for 10 minutes to allow antibody access to intracellular targets.

• Blocking: Block with 10% normal goat serum for 30 minutes at room temperature to reduce non-specific binding.

• Antibody Incubation: Apply FITC-conjugated SEC14L2 antibody at 1:50-1:200 dilution and incubate overnight at 4°C.

• Counterstaining: Use DAPI (1:1000) for nuclear staining.

• Mounting: Mount using anti-fade mounting medium to prevent photobleaching of FITC.

This protocol has been validated for detecting endogenous SEC14L2 in human cell lines such as HeLa, where SEC14L2 typically shows cytoplasmic localization with potential membrane association .

How should I design controls for SEC14L2 immunofluorescence experiments?

Proper controls are essential for interpreting SEC14L2 immunofluorescence results:

• Positive Control: Include samples known to express SEC14L2 (e.g., liver or prostate tissue sections, or PC3 cell lysates for Western validation) .

• Negative Control:

  • Primary antibody omission: Apply only buffer instead of primary antibody

  • Isotype control: Use FITC-conjugated rabbit IgG at the same concentration

  • Peptide blocking: Pre-incubate antibody with immunizing peptide

• Validation Controls:

  • Parallel Western blot to confirm specificity (expected band at 47 kDa)

  • siRNA knockdown of SEC14L2 to demonstrate specificity of staining

• Autofluorescence Control: Examine unstained samples to identify any natural fluorescence

• Signal Specificity: Test in multiple cell lines with varying SEC14L2 expression levels

These controls help distinguish true SEC14L2 signal from artifacts and ensure experimental rigor .

What are the recommended dilutions and incubation conditions for optimal SEC14L2-FITC antibody performance?

For immunofluorescence, optimal results are typically obtained when SEC14L2-FITC antibody is used at 1:133 dilution with overnight incubation at 4°C. The cellular distribution pattern (cytoplasmic with potential membrane association) should be consistent with SEC14L2's known function as a phosphatidylinositol transfer protein . Lower concentrations may require longer incubation times, while higher concentrations might increase background fluorescence.

How can SEC14L2-FITC antibodies be used to investigate the role of SEC14L2 in Wnt/Ca²⁺ signaling pathways?

SEC14L2 has been identified as a critical component in Wnt/Ca²⁺ signaling, acting as a GTPase protein that transduces signals from Frizzled to phospholipase C. To investigate this role:

• Co-localization Studies: Use SEC14L2-FITC antibodies in combination with antibodies against Frizzled receptors, Dishevelled, or PLCδ4a labeled with spectrally distinct fluorophores to visualize protein complexes.

• Translocation Experiments:

  • Treat cells with Wnt ligands and monitor SEC14L2 translocation to the plasma membrane

  • Use time-lapse imaging with SEC14L2-FITC to track real-time movement following Wnt stimulation

• FRET Analysis: Employ SEC14L2-FITC as a donor fluorophore with acceptor fluorophores conjugated to interaction partners to quantify protein-protein interactions.

• Calcium Imaging: Combine SEC14L2-FITC staining with calcium indicators to correlate SEC14L2 activity with calcium flux.

• Mutational Analysis: Compare localization patterns of wild-type versus GTPase-dead SEC14L2 mutants using SEC14L2-FITC antibodies that recognize specific domains.

These approaches can reveal how SEC14L2 forms complexes with Frizzled, Dishevelled, and PLCδ proteins, and how it regulates PIP₂ hydrolysis rather than PIP₂ transfer .

What strategies can address potential photobleaching issues when working with SEC14L2-FITC antibodies in long-term imaging experiments?

FITC is prone to photobleaching, which can limit its utility in extended imaging sessions. To overcome this:

• Anti-fade Reagents: Use mounting media containing anti-fade agents such as n-propyl gallate, p-phenylenediamine, or commercial preparations.

• Imaging Parameters Optimization:

  • Reduce exposure time and light intensity

  • Increase gain/sensitivity of detectors

  • Use neutral density filters

  • Select appropriate binning settings

• Alternative Approaches:

  • Consider sequential imaging of multiple fields rather than continuous imaging of one field

  • Use interval photography with shutter control between acquisitions

• Oxygen Scavenging Systems: Incorporate oxygen scavenger systems (e.g., glucose oxidase/catalase) to reduce reactive oxygen species generation.

• Alternative Fluorophores: For particularly challenging long-term experiments, consider requesting custom conjugation of SEC14L2 antibodies with more photostable fluorophores like Alexa Fluor 488.

• Computational Approaches: Apply deconvolution algorithms or photobleaching correction during image analysis.

For time-course experiments examining SEC14L2 dynamics in response to stimuli, balance acquisition frequency against photobleaching risk to maintain signal throughout the experimental timeline .

How can I quantify SEC14L2 expression levels using FITC-conjugated antibodies in flow cytometry?

For quantitative flow cytometric analysis of SEC14L2 expression:

• Standardization Protocol:

  • Use calibration beads with known fluorescein molecule equivalents

  • Create a standard curve relating mean fluorescence intensity (MFI) to molecules of equivalent fluorescein (MESF)

• Sample Preparation:

  • Permeabilize cells appropriately (0.1% saponin or 0.2% Triton X-100)

  • Maintain consistent cell concentration (1x10⁶ cells/mL)

  • Include compensation controls if using multiple fluorophores

• Data Acquisition Settings:

  • Establish consistent PMT voltage settings across experiments

  • Acquire sufficient events (≥10,000) for statistical reliability

• Analysis Framework:

  • Gate on viable, single cells using appropriate markers

  • Calculate net MFI by subtracting isotype control values

  • Convert to relative or absolute expression units using standard curves

• Result Validation:

  • Compare flow cytometry data with Western blot quantification

  • Analyze SEC14L2 expression across cell lines with known expression differences

This quantitative approach allows for comparison of SEC14L2 expression levels across different experimental conditions, cell types, or disease states, providing insights into its regulation and potential role in pathologies associated with altered lipid metabolism or Wnt signaling .

How should I address inconsistent staining patterns when using SEC14L2-FITC antibodies in different cell types?

Inconsistent staining patterns with SEC14L2-FITC antibodies may reflect biological variation or technical issues. To systematically address this:

• Expression Level Verification:

  • Confirm SEC14L2 expression in each cell type via RT-qPCR or Western blot

  • Consider that low expressers may require signal amplification techniques

• Fixation and Permeabilization Optimization:

  • Test multiple fixatives (4% PFA, methanol, acetone)

  • Vary permeabilization conditions (0.1-0.5% Triton X-100, saponin, digitonin)

  • Different cell types may require adjusted permeabilization due to membrane composition differences

• Antibody Accessibility Assessment:

  • Epitope masking may occur in certain cell types due to protein-protein interactions

  • Try antigen retrieval methods (heat-induced or enzymatic)

• Subcellular Localization Variations:

  • SEC14L2 localization may legitimately differ between cell types based on functional state

  • Confirm patterns with subcellular fractionation followed by Western blot

  • Use co-localization with organelle markers to characterize distribution

• Post-translational Modification Considerations:

  • SEC14L2 may undergo cell-type-specific modifications affecting epitope recognition

  • Compare results with antibodies targeting different epitopes

The reported Wnt-induced translocation of SEC14L2 to the plasma membrane suggests its localization is dynamic and may appear different depending on the activation state of signaling pathways in different cell types .

What are the key considerations when designing experiments to investigate SEC14L2's role in phosphatidylinositol transfer and signaling?

When investigating SEC14L2's functional role in phosphatidylinositol transfer and signaling:

• GTPase Activity Assessment:

  • Design experiments that distinguish between SEC14L2's PI transfer function and its GTPase activity

  • Use SEC14L2 mutants deficient in GTP binding/hydrolysis

  • Monitor PIP₂ levels using the PLCδ1-PH-GFP probe under various conditions

• Protein Complex Investigation:

  • Design co-immunoprecipitation experiments to capture SEC14L2's interactions with Frizzled, Dishevelled, and PLCδ proteins

  • Consider that SEC14L3-GFP can be co-immunoprecipitated with SEC14L3-Flag, suggesting oligomerization

  • Use proximity ligation assays to visualize protein-protein interactions in situ

• Membrane Association Dynamics:

  • Track SEC14L2 membrane association in response to Wnt stimulation

  • Employ FRAP (Fluorescence Recovery After Photobleaching) to analyze protein mobility

• Calcium Signaling Integration:

  • Correlate SEC14L2 activity with calcium release using simultaneous imaging

  • Use calcium chelators to determine dependency of observed effects

• Functional Outcome Measures:

  • In zebrafish models, convergent and extension (CE) defects can be assessed following sec14l3 depletion

  • In cell culture, monitor downstream effects on gene expression or cell behaviors

These approaches can help distinguish between SEC14L2's functions in PIP₂ hydrolysis versus PIP₂ transfer, as research indicates SEC14L2 may be required for PIP₂ hydrolysis rather than transfer .

How can I distinguish between specific and non-specific binding when using SEC14L2-FITC antibodies in tissues with high autofluorescence?

Working with tissues that exhibit high autofluorescence (like liver, where SEC14L2 is normally expressed) presents challenges for FITC-based detection. To distinguish specific signal:

• Spectral Unmixing Techniques:

  • Acquire spectral profiles of both FITC and tissue autofluorescence

  • Use linear unmixing algorithms to separate specific signal from background

  • Consider using confocal microscopes with spectral detectors

• Autofluorescence Reduction Methods:

  • Pretreat tissues with Sudan Black B (0.1-0.3%) for 10-20 minutes

  • Apply CuSO₄ (10mM in 50mM ammonium acetate) to quench autofluorescence

  • Consider photobleaching autofluorescence before antibody application

• Advanced Image Analysis:

  • Use signal-to-noise ratio calculations across multiple samples

  • Apply automated thresholding based on control samples

  • Implement machine learning algorithms to differentiate staining patterns

• Alternative Detection Strategies:

  • Consider using SEC14L2 antibodies conjugated to fluorophores with longer wavelengths (e.g., Cy3, Cy5)

  • Time-gated detection can exploit differences in fluorescence lifetime between FITC and autofluorescence

• Validation Approaches:

  • Perform RNA in situ hybridization for SEC14L2 mRNA in parallel sections

  • Use SEC14L2 knockout tissues as negative controls

  • Compare patterns across antibodies targeting different SEC14L2 epitopes

Studies have successfully used SEC14L2 antibodies in tissues with high autofluorescence, including liver and prostate, by implementing appropriate controls and optimizing detection parameters .

How might SEC14L2-FITC antibodies be used to investigate SEC14L2's role in disease processes, particularly in cancer research?

SEC14L2-FITC antibodies can provide valuable insights into SEC14L2's role in disease processes, particularly in cancer, through several approaches:

• Expression Profiling in Clinical Samples:

  • Quantify SEC14L2 expression in tumor microarrays versus matched normal tissues

  • Correlate expression with clinical parameters and patient outcomes

  • Published data indicate associations between SEC14L2 and multiple neoplasms, including prostate, breast, liver, and lung cancers

• Mechanism Investigation:

  • Examine co-localization of SEC14L2 with oncogenic signaling components

  • Study SEC14L2's involvement in lipid metabolism alterations characteristic of cancer cells

  • Investigate its interaction with Wnt signaling, which is frequently dysregulated in cancers

• Therapeutic Target Assessment:

  • Monitor changes in SEC14L2 localization and expression following treatment with anti-cancer agents

  • Evaluate effects of SEC14L2 modulation on cancer cell sensitivity to therapy

• Biomarker Development:

  • Assess SEC14L2 as a potential diagnostic or prognostic biomarker

  • Develop quantitative flow cytometry protocols for clinical specimens

• Functional Studies in Cancer Models:

  • Track SEC14L2 dynamics during cancer progression in xenograft models

  • Correlate with changes in Wnt/Ca²⁺ signaling and lipid metabolism

Research has linked SEC14L2 to multiple cancer types, with >11 publications on neoplasms, >4 on prostatic diseases, >3 on breast neoplasms, and >3 on liver neoplasms, suggesting its potential importance in cancer biology .

What methodological advances might improve SEC14L2 protein interaction studies using FITC-conjugated antibodies?

Emerging methodological advances that could enhance SEC14L2 protein interaction studies include:

• Super-Resolution Microscopy Techniques:

  • Apply STORM or PALM imaging to visualize SEC14L2 interactions at nanometer resolution

  • Use structured illumination microscopy (SIM) to improve resolution of SEC14L2 complexes

  • These techniques can reveal spatial relationships between SEC14L2 and interacting partners like Frizzled, Dishevelled, and PLCδ4a beyond the diffraction limit

• Live-Cell Interaction Monitoring:

  • Implement FRET-FLIM (Fluorescence Lifetime Imaging) to quantify interactions without intensity artifacts

  • Apply single-molecule tracking to follow individual SEC14L2 molecules and their interaction dynamics

  • Use optogenetic approaches to selectively activate SEC14L2 and observe downstream effects

• Multiplexed Detection Systems:

  • Employ cyclic immunofluorescence to detect >40 proteins in the same sample

  • Use mass cytometry with metal-tagged antibodies for high-dimensional analysis

  • Apply spectral flow cytometry to quantify multiple interaction partners simultaneously

• Proximity-Based Labeling:

  • Combine SEC14L2-FITC imaging with BioID or APEX2 proximity labeling

  • Identify transient interaction partners through spatial proteomics

• Artificial Intelligence Integration:

  • Apply deep learning algorithms to identify subtle co-localization patterns

  • Use machine learning to predict interaction networks based on imaging data

Research indicates SEC14L2 forms complexes with Frizzled, Dishevelled, and PLCδ proteins, and may oligomerize through its CARL-TRIO and GOLD domains, making these advanced methods particularly valuable for understanding its complex interaction network .

How can researchers integrate SEC14L2-FITC antibody data with genomic and proteomic datasets to gain systems-level insights?

Integrating SEC14L2-FITC antibody data with multi-omics approaches enables systems-level understanding:

• Spatial Transcriptomics Correlation:

  • Combine SEC14L2 immunofluorescence imaging with spatial transcriptomics

  • Map SEC14L2 protein distribution against transcriptional landscapes

  • Identify spatial regions where SEC14L2 protein presence correlates with specific gene expression patterns

• Proteogenomic Integration:

  • Correlate SEC14L2 localization patterns with phosphoproteomic data

  • Integrate with genomic data to identify mutations affecting SEC14L2 localization or expression

  • Map SEC14L2 interactions against protein interaction networks

• Multi-modal Data Fusion Approaches:

  • Develop computational methods to integrate imaging, genomic, and proteomic data

  • Apply tensor factorization or similar mathematical frameworks to identify patterns across data types

  • Implement graph-based methods to place SEC14L2 in functional networks

• Pathway Analysis Enhancement:

  • Use SEC14L2 localization data to refine pathway models of Wnt signaling

  • Incorporate information about SEC14L2's role in phosphatidylinositol metabolism

  • Create dynamic models incorporating temporal SEC14L2 behavior

• Disease Association Mapping:

  • Correlate SEC14L2 expression and localization with disease-associated genomic variants

  • Identify potential disease mechanisms through integration with clinical data

Given SEC14L2's roles in both lipid metabolism and Wnt signaling, integrated approaches are particularly valuable for understanding how these functions intersect in normal physiology and disease .