LGALS14 Antibody

What is LGALS14 and what are its key biological functions?

LGALS14, also known as Galectin-14, is a member of the galectin family of beta-galactoside-binding proteins. Its primary functions include binding to beta-galactoside and lactose molecules within cellular environments . Notably, LGALS14 serves as a strong inducer of T-cell apoptosis, suggesting an important immunomodulatory role . This protein is structurally related to other galectins but possesses unique binding properties and tissue distribution patterns that distinguish it within this protein family.

What alternative nomenclature is used for LGALS14 in scientific literature?

Researchers should be aware of multiple designations when searching literature for LGALS14. Common alternative names include:

• Placental protein 13-like (PPL13)

• Charcot-Leyden crystal protein 2 (CLC2)

• Galectin-14 (Gal-14)

Using these alternative terms during literature searches ensures comprehensive coverage of relevant research.

What is the expression pattern of LGALS14 in normal and pathological tissues?

LGALS14 demonstrates a relatively restricted expression pattern, with notable expression in placental tissue as evidenced by its detection in human placenta lysates . Research indicates that LGALS14 is also expressed in ovarian cancer cells, particularly showing elevated expression in high-grade serous adenocarcinoma (HGSA) . Interestingly, while mRNA expression is readily detectable in certain cancer cell lines, protein-level detection can be challenging, suggesting potential post-transcriptional regulation mechanisms .

What criteria should guide selection of an appropriate anti-LGALS14 antibody?

When selecting an anti-LGALS14 antibody, researchers should consider:

• Antibody specificity: Verify cross-reactivity profiles, particularly with other galectin family members. For example, research has shown that some commercial antibodies exhibit low but detectable cross-reactivity with human galectin-7 and galectin-1 .

• Validated applications: Confirm the antibody has been validated for your specific application (e.g., Western blot, immunofluorescence, flow cytometry) .

• Species reactivity: Ensure compatibility with your experimental model. Available antibodies have demonstrated reactivity with human and mouse samples .

• Clone type: Consider whether monoclonal or polyclonal antibodies are more appropriate for your research question. For instance, the EPR9665 clone (ab150427 from Abcam) is a rabbit recombinant monoclonal antibody validated for several applications .

How can researchers validate the specificity of anti-LGALS14 antibodies?

Validation should include:

• Positive control samples: Human placenta lysates have been successfully used as positive controls for LGALS14 detection . Recombinant human LGALS14 protein can also serve as a reliable positive control .

• Comparison of multiple antibodies: When possible, compare results from different antibodies targeting different epitopes of LGALS14. Research has shown variability in specificity between commercial antibodies .

• Secondary antibody-only controls: Include controls omitting the primary antibody to assess potential non-specific binding of secondary antibodies .

• Knockdown/knockout validation: If feasible, use genetic approaches to reduce endogenous LGALS14 expression for validation of antibody specificity.

What are the optimal conditions for Western blot detection of LGALS14?

For successful Western blot detection of LGALS14:

• Sample preparation: Human placenta lysates have shown successful detection of LGALS14. Mouse placenta lysates can also be used .

• Antibody dilution: For the EPR9665 clone, a 1/1000 dilution has been validated for Western blot applications .

• Protein loading: 10-15 μg of total protein per lane is recommended based on successful detection protocols .

• Expected molecular weight: The predicted band size for LGALS14 is 16 kDa, which corresponds to observed bands in validated Western blots .

• Secondary antibody selection: Goat anti-rabbit HRP at 1/2000 dilution has been successfully used with rabbit primary antibodies against LGALS14 .

What protocol is recommended for immunofluorescence studies of LGALS14?

For immunocytochemistry/immunofluorescence applications:

• Cell fixation: 4% paraformaldehyde fixation has been validated for LGALS14 detection .

• Antibody dilution: For the EPR9665 clone, a 1/100 dilution (10 μg/ml) is recommended .

• Cell models: JAR cells (human placenta choriocarcinoma epithelial cells) have been successfully used for LGALS14 immunofluorescence studies .

• Secondary detection: Goat anti-rabbit IgG coupled to Alexa Fluor 488 at 1/1000 dilution (2 μg/ml) provides effective visualization .

• Counterstaining: DAPI for nuclear visualization and possibly additional markers such as alpha-tubulin for cytoskeletal reference .

How can LGALS14 be detected by flow cytometry?

For intracellular flow cytometry detection of LGALS14:

• Cell preparation: Fix cells with 4% paraformaldehyde followed by permeabilization with 90% methanol .

• Antibody concentration: For the EPR9665 clone, a 1/20 dilution (10 μg/ml) has been validated .

• Cell type: JAR cells have shown detectable LGALS14 expression by flow cytometry .

• Controls: Include isotype controls (rabbit monoclonal IgG) and unlabeled controls (cells without primary and secondary antibody incubation) .

What is the significance of LGALS14 expression in ovarian cancer?

LGALS14 has emerged as a protein of interest in ovarian cancer research based on several observations:

• Survival correlation: High expression of LGALS14 mRNA in ovarian cancer is associated with shorter patient survival (p = 0.016), suggesting potential prognostic value .

• Cancer subtype association: LGALS14 is preferentially expressed in high-grade serous adenocarcinoma (HGSA), the most aggressive subtype of ovarian cancer .

• Differential expression: LGALS14 expression is significantly higher in epithelial ovarian cancer (EOC) tissues compared to normal tissues (p = 0.03) .

• Treatment resistance: There is a trend toward differential expression between treatment-sensitive and treatment-resistant cases, though this observation was just above the threshold of statistical significance (p = 0.1) .

What mechanisms might explain the role of LGALS14 in cancer progression?

Research suggests several potential mechanisms:

• Apoptotic regulation: When expressed in HEK-293 cells, LGALS14 increases both basal apoptosis and apoptosis following exposure to low doses of etoposide, indicating a role in cell death processes .

• Genomic alterations: LGALS14 exhibits a higher percentage of genetic amplification in epithelial ovarian cancer compared to other galectin family members .

• Expression discrepancy: While mRNA expression is detectable in ovarian cancer cell lines, protein-level detection is challenging, suggesting complex post-transcriptional regulation .

Why might researchers encounter difficulties detecting LGALS14 protein despite detectable mRNA expression?

This discrepancy has been documented in ovarian cancer research and may result from:

• Low protein abundance: Cells may express very low levels of LGALS14 protein despite readily detectable mRNA .

• Protein instability: LGALS14 protein may be rapidly degraded, particularly if it induces apoptosis when expressed at high levels .

• Post-transcriptional regulation: miRNAs or other regulatory mechanisms may inhibit translation of LGALS14 mRNA.

• Antibody sensitivity limitations: Current antibodies may lack sufficient sensitivity to detect very low protein levels that are nonetheless biologically significant .

What strategies can improve LGALS14 protein detection?

To enhance detection sensitivity:

• Protein enrichment: Consider using immunoprecipitation to concentrate LGALS14 protein before Western blotting.

• Alternative detection methods: More sensitive detection systems such as chemiluminescent substrates with extended exposure times may help.

• Proteasome inhibition: If rapid degradation is suspected, treating cells with proteasome inhibitors before protein extraction may increase detectable levels.

• Positive controls: Always include known positive controls such as placenta lysates or recombinant LGALS14 protein .

How does LGALS14 compare functionally to other galectin family members?

While research on LGALS14 is still in its early stages compared to more extensively studied galectins:

• Shared functions: Like other galectins, LGALS14 binds beta-galactoside and lactose .

• Unique tissue distribution: LGALS14 shows a more restricted expression pattern than widely expressed galectins like galectin-1 and galectin-3 .

• Genomic alterations in cancer: LGALS14 harbors more genomic alterations in ovarian cancer patients than some well-characterized family members like LGALS1 and LGALS3 .

• Apoptotic activity: LGALS14 functions as a strong T-cell apoptosis inducer and increases basal apoptosis when expressed in HEK-293 cells, suggesting a prominent role in cell death regulation .

What experimental models are most appropriate for studying LGALS14 function?

Based on current research, promising experimental models include:

• Placental models: Given its expression in placenta, models such as JAR cells (human placenta choriocarcinoma) and placental tissue explants are appropriate .

• Ovarian cancer models: High-grade serous adenocarcinoma cell lines, particularly OVCAR-3, express detectable levels of LGALS14 mRNA .

• HEK-293 transfection models: Transfection of LGALS14 into HEK-293 cells allows for functional studies, particularly regarding its role in apoptosis .

• Mouse models: Mouse placenta expresses LGALS14, providing potential for in vivo studies .