FAM131A Antibody

What is FAM131A and what cell types express this protein?

FAM131A is a protein encoded by the FAM131A gene (also known as C3orf40). Based on single-cell sequencing data from the human lung atlas, FAM131A is strongly expressed in airway epithelium, particularly in ciliated cells. It also shows substantial expression in fibroblasts and certain neutrophil subsets . Immunohistochemistry studies have confirmed protein expression in airway epithelial cells, infiltrated immune cells, stromal cells, and smooth muscle cells, suggesting multiple roles in lung physiology .

The expression pattern varies significantly between healthy individuals and those with respiratory conditions. In particular, immunohistochemistry studies have demonstrated that FAM131A protein expression is significantly weaker in the airways of COPD patients compared to non-COPD controls, suggesting potential involvement in disease pathogenesis .

What are the available FAM131A antibodies and their applications?

Several FAM131A antibodies are commercially available for research applications. The following table summarizes key antibodies and their specifications:

Most FAM131A antibodies are suitable for multiple applications with specific recommended dilutions:

• Immunohistochemistry (IHC): Typically at dilutions of 1:20-1:500

• Western Blotting (WB): Typically at concentrations of 0.04-0.4 μg/mL

• Immunofluorescence (IF): Typically at concentrations of 0.25-2 μg/mL

• ELISA: Following manufacturer's recommended protocols

What are the optimal protocols for FAM131A detection by immunohistochemistry?

Based on published research methodologies, the following protocol is recommended for FAM131A detection by immunohistochemistry:

• Sample preparation: Embed lung tissue in paraffin and cut into three-μm thick sections

• Antigen retrieval: Perform with 10 mM citrate buffer at pH 6.0

• Blocking: Apply avidin/biotin blocking using an Avidin/Biotin blocking kit

• Primary antibody application: Use FAM131A-specific antibody (e.g., 55401-1-AP, Proteintech) at dilutions between 1:20-1:200

• Secondary antibody: Apply appropriate HRP-conjugated secondary antibody (e.g., Goat Anti-Rabbit Immunoglobulins/HRP)

• Tertiary antibody (if applicable): Use Streptavidin/HRP

• Visualization: Develop using an appropriate detection system

For optimal results, researchers should validate this protocol with their specific tissue types and antibodies, as minor modifications may be necessary depending on sample characteristics and antibody properties.

How should FAM131A antibodies be stored and handled?

For maximum stability and performance of FAM131A antibodies:

• Storage temperature: Store at -20°C or -80°C according to manufacturer's recommendations

• Avoid repeated freeze-thaw cycles as noted in storage recommendations

• Most antibodies are provided in buffered aqueous glycerol solution (typically 50% glycerol, 0.01M PBS, pH 7.4)

• When working with antibodies containing preservatives like ProClin (0.03%), handle with appropriate precautions as these are classified as hazardous substances requiring trained staff handling

• For shipping between facilities, transport on wet ice

• Upon receipt, promptly store according to manufacturer guidelines to maintain antibody integrity

Proper storage and handling are critical for maintaining antibody performance and extending shelf-life.

How does FAM131A expression differ between COPD patients and healthy controls?

Research has revealed significant differences in FAM131A expression between COPD patients and non-COPD controls:

These findings suggest that reduced FAM131A expression may contribute to COPD pathogenesis or represent a consequence of disease processes. The differential response to CSE between COPD and non-COPD cells indicates potential disease-specific regulatory mechanisms affecting FAM131A expression.

What is the effect of cigarette smoke extract on FAM131A expression?

The response of FAM131A expression to cigarette smoke extract (CSE) exposure appears to be disease-state dependent:

• In COPD-derived primary human airway epithelial cells:

  • 24-hour exposure to 20% CSE significantly decreased FAM131A protein levels

• In non-COPD control-derived airway epithelial cells:

  • No significant change in FAM131A expression was observed after CSE exposure at the same concentration

• In bronchial epithelial cell line 16HBE14o- with experimentally overexpressed FAM131A:

  • Exposure to 10% CSE did not affect the overexpression of FAM131A

This differential response suggests COPD-specific cellular mechanisms regulating FAM131A expression in response to cigarette smoke components. These findings point to a potential role for FAM131A in COPD susceptibility or disease progression, particularly in relation to cigarette smoke exposure, which is the primary risk factor for COPD development.

What is the role of FAM131A in epithelial barrier function?

Research using overexpression models has revealed important functions of FAM131A in epithelial barrier formation and integrity:

• Barrier formation kinetics: In 16HBE14o- cells overexpressing FAM131A, epithelial resistance (measured by electric cell-substrate impedance sensing at 400 Hz) developed significantly more rapidly, indicating enhanced formation of cell-cell contacts .

• Junctional protein expression: FAM131A overexpression correlates with higher E-cadherin expression, suggesting a role in adherens junction formation or stability .

• Inflammatory response modulation: Cells overexpressing FAM131A showed reduced cigarette smoke extract-induced CXCL8 levels, indicating an attenuated pro-inflammatory response to environmental insults .

These findings suggest that FAM131A may protect airway epithelial barrier integrity by promoting cell-cell contact formation and reducing inflammatory responses to harmful stimuli. The reduced expression of FAM131A in COPD airway epithelium may therefore contribute to impaired barrier function and enhanced inflammation observed in this disease.

How does FAM131A relate to β-catenin and E-cadherin expression?

FAM131A appears to interact with key components of the adherens junction complex:

• β-catenin regulation: Previous research has demonstrated that FAM131A can regulate β-catenin stability, a critical component of both adherens junctions and Wnt signaling pathways . This suggests FAM131A may influence these essential cellular processes.

• E-cadherin relationship: Overexpression of FAM131A in bronchial epithelial cells results in higher E-cadherin expression . E-cadherin is the primary adhesion molecule in epithelial adherens junctions, essential for maintaining epithelial integrity.

• Functional implications: The interaction between FAM131A, β-catenin, and E-cadherin suggests that FAM131A may promote epithelial barrier function by enhancing adherens junction formation or stability, potentially through stabilization of protein complexes at cell-cell junctions .

These molecular interactions provide mechanistic insights into how decreased FAM131A expression in COPD airway epithelium might contribute to impaired barrier function, a hallmark of airway disease.

What methodological challenges exist in detecting FAM131A in different tissue types?

Researchers face several technical challenges when detecting FAM131A across different tissue types:

• Expression heterogeneity: FAM131A expression varies considerably between cell types within the lung, with strongest expression in airway epithelium (particularly ciliated cells) and weaker expression in other cell types .

• Disease-related expression changes: In conditions like COPD, FAM131A expression is significantly reduced in specific cell types (airway epithelium), requiring higher sensitivity detection methods .

• Antibody specificity concerns: Ensuring antibody specificity is critical, particularly when examining tissues with complex cellular composition. Validation using multiple antibodies targeting different epitopes is recommended .

• Detection method sensitivity variations: Different detection methods (IHC, IF, WB) have varying sensitivities for FAM131A detection. Researchers should optimize protocols based on the expected expression level in their tissue of interest .

• Background signal management: In complex tissues, distinguishing specific FAM131A signal from background can be challenging, requiring careful optimization of blocking and washing steps.

To overcome these challenges, researchers should consider using multiple detection methods, rigorously validated antibodies, and appropriate positive and negative controls.

How should experiments be designed to study FAM131A in airway epithelial cells?

When designing experiments to study FAM131A in airway epithelial cells, researchers should consider:

• Cell model selection:

  • Primary human airway epithelial cells (AECs) from both COPD patients and healthy controls for disease relevance

  • Established cell lines like 16HBE14o- that have been validated to model primary AECs for mechanistic studies

  • Air-liquid interface cultures for more physiologically relevant models of the airway epithelium

• Experimental conditions:

  • Baseline conditions to assess constitutive expression

  • Cigarette smoke extract (CSE) exposure (typically 10-20%) to model smoking effects

  • Time course experiments (24-72 hours) to capture dynamic changes in expression and function

• Analytical approaches:

  • Protein expression: Western blotting (0.04-0.4 μg/mL antibody concentration), immunofluorescence (0.25-2 μg/mL), immunohistochemistry (1:20-1:500 dilution)

  • Functional assays: Epithelial barrier function using Electric Cell-substrate Impedance Sensing (ECIS) at 400 Hz for cell-cell contacts

  • Inflammatory response: ELISA for CXCL8 or other inflammatory mediators

• Manipulation approaches:

  • Overexpression using plasmid transfection

  • siRNA or CRISPR for knockdown/knockout studies

  • Rescue experiments to confirm specificity of observed effects

A comprehensive experimental design should include appropriate controls, sufficient biological replicates (n≥3), and statistical analysis appropriate for the data distribution (e.g., Wilcoxon signed-rank test for within-group comparisons, Mann-Whitney test for between-group differences) .

What controls should be included when using FAM131A antibodies?

When using FAM131A antibodies in research, the following controls should be included:

• Positive controls:

  • Tissues known to express FAM131A (e.g., normal airway epithelium)

  • Cell lines with confirmed FAM131A expression

  • Recombinant FAM131A protein (when available)

  • Cells transfected with FAM131A expression vectors

• Negative controls:

  • Isotype control antibodies to assess non-specific binding

  • Secondary antibody-only controls to detect background staining

  • Pre-absorption with immunizing peptide to confirm specificity

• Technical controls for specific applications:

  • Loading controls for Western blotting (β-actin, GAPDH)

  • Nuclear counterstains (DAPI, Hoechst) for immunofluorescence

  • Tissue-specific internal controls for immunohistochemistry

• Experimental controls:

  • Vehicle controls for treatments (e.g., media control for CSE exposure)

  • Empty vector controls for overexpression studies

  • Time-matched controls for time-course experiments

Including these controls helps ensure the specificity and reliability of results obtained with FAM131A antibodies across different experimental conditions and techniques.

How can researchers validate the specificity of FAM131A antibodies?

Validating antibody specificity is crucial for reliable research outcomes. For FAM131A antibodies, researchers should consider:

• Multiple antibody validation:

  • Use antibodies from different manufacturers targeting different epitopes (e.g., ABIN7165390 targeting AA 1-281 and others targeting C-terminal regions)

  • Compare staining/detection patterns across antibodies

  • Confirm consistent expression patterns across techniques (WB, IHC, IF)

• Molecular validation:

  • Correlate protein detection with mRNA expression (RT-PCR, RNA-seq)

  • Use gene silencing (siRNA/shRNA) or CRISPR knockout to confirm signal specificity

  • Perform overexpression studies to confirm increased signal intensity

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide (e.g., recombinant human FAM131A protein)

  • Observe elimination or reduction of specific signal

• Cross-species validation:

  • Test antibody in species with high sequence homology (e.g., HPA042800 is reactive with human, mouse, and rat samples)

  • Confirm expected expression patterns across species

• Application-specific validation:

  • For IHC: Include tissue with known expression patterns

  • For WB: Confirm correct molecular weight and single band specificity

  • For IF: Co-localize with known interaction partners (e.g., E-cadherin)

What are the recommended techniques for quantifying changes in FAM131A expression?

Accurate quantification of FAM131A expression is essential for meaningful comparisons between experimental conditions. Several methods are recommended:

• Western blotting quantification:

  • Normalize FAM131A band intensity to loading controls (β-actin, GAPDH)

  • Use digital imaging and analysis software (ImageJ, Image Lab)

  • Present relative expression as fold-change from baseline/control

• Immunohistochemistry quantification:

  • Use digital pathology software for automated scoring

  • Assess both staining intensity and percentage of positive cells

  • Develop scoring systems (e.g., H-score)

  • Blind scoring by multiple observers to reduce bias

• Immunofluorescence quantification:

  • Measure mean fluorescence intensity within defined regions of interest

  • Analyze co-localization with other markers using Pearson's correlation

  • Use nuclear counterstains for cell identification and normalization

• Statistical analysis approaches:

  • For non-normally distributed data: Use non-parametric tests (Wilcoxon signed-rank, Mann-Whitney)

  • For normally distributed data: Apply parametric tests (t-test, ANOVA)

  • Consider p < 0.05 as statistically significant

  • Present data as median with interquartile range or mean ± standard deviation as appropriate

Consistency in quantification methods across experiments facilitates reliable comparisons and reproducibility of research findings.

What methodologies can assess functional consequences of FAM131A manipulation?

To evaluate the functional impact of FAM131A expression changes, researchers should consider these methodologies:

• Epithelial barrier assessment:

  • Electric Cell-substrate Impedance Sensing (ECIS): Monitor resistance at 400 Hz for cell-cell contacts and capacitance at 32,000 Hz for cell-substrate contacts

  • Transepithelial electrical resistance (TEER) measurements

  • Paracellular permeability assays using fluorescent tracers

• Cell-cell adhesion analysis:

  • Immunofluorescence for junctional proteins (E-cadherin, β-catenin)

  • Western blotting for quantitative assessment of junctional protein expression

  • Calcium switch assays to assess junction formation kinetics

• Inflammatory response evaluation:

  • ELISA for pro-inflammatory cytokines/chemokines (e.g., CXCL8)

  • Multiplex cytokine assays for comprehensive inflammatory profile

  • NF-κB activation assays (reporter systems, nuclear translocation)

• Cell functionality tests:

  • Wound healing/scratch assays to assess repair capacity

  • Proliferation assays (MTT, BrdU incorporation)

  • Apoptosis assessment (Annexin V, TUNEL)

• Pathway analysis:

  • β-catenin nuclear translocation by immunofluorescence

  • TCF/LEF reporter assays for Wnt pathway activation

  • Co-immunoprecipitation to assess protein-protein interactions

These complementary approaches provide a comprehensive assessment of FAM131A's role in epithelial cell biology and potential involvement in pathological conditions like COPD.

What are common issues when detecting FAM131A and how can they be resolved?

Researchers may encounter several challenges when detecting FAM131A. Here are common issues and their solutions:

• Weak or absent signal in immunostaining:

  • Optimize antigen retrieval: Test different buffers (citrate pH 6.0, EDTA pH 9.0) and times

  • Increase antibody concentration: Try higher concentrations within recommended ranges (1:20-1:200 for IHC)

  • Extend incubation time: Consider overnight primary antibody incubation at 4°C

  • Use signal amplification systems: Consider tyramide signal amplification for low-abundance targets

• High background in immunostaining:

  • Optimize blocking: Increase blocking time or use different blocking agents

  • Reduce antibody concentration: Test lower concentrations within recommended ranges

  • Increase washing: Add additional wash steps or extend wash times

  • Use more specific detection systems: Consider polymer-based detection instead of avidin-biotin

• Multiple bands in Western blotting:

  • Optimize lysis conditions: Test different buffer compositions

  • Adjust running conditions: Use gradient gels for better separation

  • Increase antibody specificity: Pre-absorb with immunizing peptide

  • Confirm identity: Consider using knockout/knockdown controls

• Inconsistent results between experiments:

  • Standardize protocols: Document and follow consistent procedures

  • Control for cell density/confluence: Seed cells at consistent densities

  • Document antibody lots: Different lots may have different specificities

  • Include positive controls: Use samples with known expression in every experiment

These troubleshooting approaches can significantly improve detection reliability and experimental reproducibility.

How can researchers optimize FAM131A antibody-based assays for specific tissue types?

Optimization strategies for FAM131A detection in specific tissue types include:

• For lung tissue immunohistochemistry:

  • Fixation optimization: Test different fixation durations (12-24h)

  • Section thickness: Optimal thickness is typically 3μm for lung tissue

  • Antigen retrieval: 10mM citrate buffer at pH 6.0 has been successfully used

  • Antibody dilution: Start with 1:50-1:200 dilution range and optimize

• For cultured airway epithelial cells:

  • Fixation method: 4% paraformaldehyde for 10-20 minutes

  • Permeabilization: 0.1-0.3% Triton X-100 for intracellular targets

  • Blocking: 5% normal serum from the species of secondary antibody

  • Co-staining: Consider dual staining with epithelial markers (E-cadherin)

• For flow cytometry:

  • Cell preparation: Gentle enzymatic dissociation to preserve surface antigens

  • Antibody concentration: Titrate to determine optimal concentration

  • Controls: Include fluorescence minus one (FMO) controls

  • Viability dye: Include to eliminate dead cell artifacts

• For co-localization studies:

  • Sequential staining: May be necessary to avoid cross-reactivity

  • Spectral separation: Choose fluorophores with minimal spectral overlap

  • Controls: Include single-stained controls for compensation

  • Image acquisition: Use appropriate filters and sequential scanning

Tissue-specific optimization is essential as FAM131A expression varies significantly between cell types, with highest expression in airway epithelium and lower expression in other lung cells .

What are promising areas for future research on FAM131A?

Based on current knowledge, several promising research directions for FAM131A include:

• Mechanistic studies:

  • Detailed investigation of FAM131A interactions with β-catenin and E-cadherin

  • Elucidation of signaling pathways regulated by FAM131A

  • Identification of post-translational modifications affecting FAM131A function

• Disease relevance beyond COPD:

  • Exploration of FAM131A's role in other respiratory diseases (asthma, pulmonary fibrosis)

  • Investigation of FAM131A in non-respiratory epithelial barriers

  • Assessment of FAM131A in cancer progression (given its interaction with β-catenin)

• Therapeutic potential:

  • Development of approaches to restore FAM131A expression in COPD

  • Investigation of FAM131A as a biomarker for epithelial dysfunction

  • Exploration of FAM131A-targeted therapies to enhance barrier function

• Advanced models:

  • Development of FAM131A knockout or transgenic animal models

  • Application of lung-on-chip technology to study FAM131A in complex microenvironments

  • Use of patient-derived organoids to assess personalized responses

• Clinical correlations:

  • Large-scale studies correlating FAM131A expression with disease severity

  • Longitudinal studies tracking FAM131A expression over disease progression

  • Correlation with clinical outcomes and treatment responses

These research directions could significantly advance our understanding of FAM131A's biological functions and therapeutic potential.