FAR2 Antibody

What is FAR2 and what biological functions has it been associated with?

FAR2 (also known as MLSTD1 or Male sterility domain-containing protein 1) is a reductase enzyme involved in the conversion of fatty acids to fatty alcohols . Research has demonstrated its involvement in several pathological conditions. FAR2 expression is significantly associated with kidney diseases including diabetic nephropathy, lupus nephritis, and IgA nephropathy . The gene plays a crucial role in mesangial matrix expansion (MME), a key feature of chronic kidney disease progression. Mechanistically, FAR2 can mediate de novo platelet-activating factor synthesis, which may contribute to its role in disease development . In addition, recent studies suggest that FFAR2 (Free Fatty Acid Receptor 2) signaling antagonizes TLR2- and TLR3-induced lung cancer progression via suppression of the cAMP-AMPK-TAK1 signaling axis for NF-κB activation .

How does genetic variation in FAR2 impact its expression and function?

Genetic variation in the FAR2 gene has been linked to differential expression and subsequent pathological outcomes. A notable example is a 9-base pair sequence located in the 5′-untranslated region (UTR) of the gene that is present in mouse inbred strains with mesangial matrix expansion (MME) but absent in most strains without MME . This variation correlates with changes in FAR2 expression levels. Research has shown that knockdown of Far2 in mice significantly delays the progression of mesangial matrix expansion, extending the disease-free period by at least six months (equivalent to approximately 15 years in human terms) . Understanding these genetic determinants of FAR2 expression provides valuable insights for researchers studying disease mechanisms and potential therapeutic interventions.

What critical specifications should researchers evaluate when selecting a FAR2 antibody?

When selecting a FAR2 antibody, researchers should consider multiple specifications:

• Immunogen design: The specific region of FAR2 targeted by the antibody significantly impacts its utility. For example, some commercially available antibodies are generated against C-terminal regions of human FAR2 (peptide sequence: WSTYNTEMLMSELSPEDQRVFNFDVRQLNWLEYIENYVLGVKKYLLKEDM) , while others target internal residues .

• Host species and clonality: Most available FAR2 antibodies are rabbit polyclonal antibodies , which offer broad epitope recognition but may have batch-to-batch variability.

• Validated applications: Verify the antibody has been validated for your specific application. Current FAR2 antibodies have been validated primarily for Western blot (WB) and immunohistochemistry (IHC) .

• Species reactivity: Confirm the antibody recognizes FAR2 from your species of interest. Available antibodies primarily target human FAR2 , though cross-reactivity with other species may occur.

• Formulation and concentration: Antibodies come in various formulations, including BSA-free options (0.5 mg/ml) and glycerol-containing formulations (2.2 mg/ml) .

What experimental controls are essential for validating FAR2 antibodies?

Proper validation of FAR2 antibodies requires rigorous controls:

• Positive controls: Use tissues or cell lines with known FAR2 expression. Jurkat cell lysates and human fetal liver tissue have been validated as positive controls for Western blotting of FAR2.

• Negative controls: Include FAR2 knockout or knockdown samples. The KOMP2 program has generated FAR2 knockout mice (Far2^tm2a(KOMP)Wtsi/2J) that can provide tissues for negative control validation .

• Loading controls: For Western blots, include appropriate housekeeping proteins to normalize expression data.

• Antibody dilution series: Test a range of antibody concentrations to determine optimal signal-to-noise ratio. Recommended dilutions range from 0.2-1 μg/ml to 1/1000 depending on the specific antibody and application.

• Secondary antibody-only controls: Include samples treated only with secondary antibody to identify non-specific binding.

What are the optimized conditions for Western blotting with FAR2 antibodies?

Based on validated protocols, the following conditions are recommended for Western blotting with FAR2 antibodies:

• Sample preparation: Use 40 μg of total protein lysate per lane . Tissues with confirmed FAR2 expression include fetal liver, brain, and eyelid .

• Gel electrophoresis: 6% SDS-PAGE gels have been successfully used for FAR2 detection .

• Primary antibody incubation: Dilute antibodies appropriately (1/1000 dilution has been validated) . Incubate overnight at 4°C for optimal results.

• Secondary antibody: Anti-rabbit IgG conjugated to HRP at 1/8000 dilution has proven effective .

• Detection: Exposure times of approximately 40 seconds have yielded clear bands in chemiluminescence detection systems .

• Expected results: The antibody should detect endogenous levels of total FAR2 protein at the expected molecular weight.

What protocol modifications are needed for successful immunohistochemistry with FAR2 antibodies?

For immunohistochemical detection of FAR2:

• Tissue preparation: Paraffin-embedded tissue sections have been successfully used. Thyroid cancer tissue has been validated for FAR2 IHC .

• Antigen retrieval: Though specific methods aren't detailed in the search results, heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically recommended for similar targets.

• Antibody dilution: A 1/50 dilution has been validated for IHC applications .

• Detection system: While not explicitly mentioned in the search results, standard avidin-biotin or polymer-based detection systems compatible with rabbit primary antibodies would be appropriate.

• Controls: Include positive control tissues alongside experimental samples to confirm staining specificity.

How can FAR2 antibodies be utilized to investigate kidney disease mechanisms?

FAR2 antibodies offer valuable tools for investigating kidney disease mechanisms:

• Expression profiling: Analyze FAR2 protein levels across different kidney disease types. Research has established associations between FAR2 expression and diabetic nephropathy, lupus nephritis, and IgA nephropathy .

• Co-localization studies: Combine FAR2 antibodies with markers of mesangial matrix components to analyze spatial relationships in kidney tissue.

• Disease progression models: Monitor FAR2 expression changes during disease progression using serial samples and correlate with clinical parameters.

• Mechanistic investigations: Study the relationship between FAR2 and platelet-activating factor (PAF) synthesis. Research has demonstrated that FAR2 is capable of mediating de novo PAF synthesis in vitro , suggesting a mechanistic link to kidney disease.

• Therapeutic response assessment: Evaluate changes in FAR2 expression following experimental therapies to assess potential intervention efficacy.

What approaches can researchers use to study FAR2's role in cancer biology?

Research into FAR2's cancer-related functions can be advanced through:

• Expression analysis: Compare FAR2 levels between tumor and adjacent normal tissues. TCGA data shows significant down-regulation of FFAR2 in lung cancer .

• Signaling pathway interactions: Investigate FAR2's relationship with cancer-relevant pathways. FFAR2 has been shown to antagonize TLR2- and TLR3-induced lung cancer progression via the cAMP-AMPK-TAK1 signaling axis for NF-κB activation .

• Functional studies: Assess the impact of FAR2 modulation on cancer cell phenotypes. FFAR2KO A549 and FFAR2KO H1299 human lung cancer cells showed marked increases in cell migration, invasion, and colony formation .

• Cytokine profiling: Measure changes in cancer-related cytokines with FAR2 modulation. FFAR2 knockout has been associated with elevations in CCL2, IL-6, and MMP2 production .

• Therapeutic targets: Investigate FAR2 agonists as potential cancer therapeutics. Propionate (an FFAR2 agonist) significantly inhibited lung cancer migration, invasion, and colony formation induced by TLR2 or TLR3 .

How should researchers address unexpected or contradictory results when working with FAR2 antibodies?

When confronted with unexpected results:

• Antibody validation: Re-validate antibody specificity using positive and negative controls. Consider using multiple antibodies targeting different FAR2 epitopes.

• Sample quality assessment: Verify protein integrity through total protein staining or housekeeping proteins.

• Technical parameters: Optimize blocking conditions, antibody concentrations, and incubation times. For FAR2 antibodies, recommended concentrations range from 0.2-1 μg/ml for Western blot .

• Protein-mRNA correlation analysis: Compare protein data with mRNA expression using primers designed to span exon junctions (e.g., between exons 9 and 10 for FAR2) .

• Literature comparison: Consider whether your experimental context differs from published work. FAR2's diverse functions in different tissues may lead to context-dependent results.

What are the key considerations when interpreting FAR2 expression data across different experimental systems?

Interpreting FAR2 data requires consideration of several factors:

• Tissue-specific expression patterns: FAR2 expression varies across tissues, with documented expression in brain, eyelid, liver, and kidney tissues .

• Genetic background effects: Consider strain-specific genetic variations, such as the 9-bp sequence in the 5′-UTR associated with differential FAR2 expression in mice .

• Disease context: FAR2's role differs across pathological conditions. It's associated with mesangial matrix expansion in kidney disease and appears to counteract cancer progression in lung cancer models .

• Protein vs. mRNA correlation: Verify concordance between protein and transcript levels, as post-transcriptional regulation may affect FAR2.

• Antibody characteristics: Different antibodies target different epitopes, potentially yielding varied results depending on protein conformation, modifications, or interactions.

How might researchers investigate the transcriptional regulation of FAR2?

To study FAR2 transcriptional regulation:

• Transcription factor studies: Investigate NKX3.2, which has been identified as a transcription factor driving FAR2 expression .

• Promoter analysis: Characterize the FAR2 promoter region and identify regulatory elements, particularly focusing on the 5′-UTR where the 9-bp sequence variation associated with differential expression is located .

• Epigenetic regulation: Examine DNA methylation and histone modifications at the FAR2 locus across different tissues and disease states.

• Gene expression correlation: Analyze co-expression networks to identify other genes regulated alongside FAR2 that may share regulatory mechanisms.

• Transcriptional inhibitor studies: Use specific inhibitors of suspected transcription factors to confirm regulatory relationships.

What future directions might expand the therapeutic potential of targeting FAR2?

Emerging therapeutic applications include:

• Agonist development: Design and test FAR2 agonists for cancer therapy. Propionate, an FFAR2 agonist, has shown promising anti-cancer effects .

• Kidney disease interventions: Develop FAR2 inhibitors for kidney disease, based on findings that FAR2 knockdown delays mesangial matrix expansion .

• Rational antibody design: Apply kinetically controlled somatic hypermutation approaches to develop therapeutic antibodies targeting FAR2 .

• Combination therapies: Investigate FAR2-targeting agents in combination with established therapies for kidney disease or cancer.

• Biomarker development: Validate FAR2 as a diagnostic or prognostic biomarker for kidney diseases or certain cancers based on its differential expression in these conditions .