FAM13A Antibody

What is FAM13A and why is it significant for research?

FAM13A (Family with Sequence Similarity 13 Member A) is a gene that exhibits high sequence conservation across species and has been implicated in several important biological processes. It has gained significant research interest due to its associations with chronic obstructive pulmonary disease (COPD), non-small cell lung cancer (NSCLC), adipocyte function, and metabolic traits identified through genome-wide association studies (GWAS) . FAM13A contains a RhoGAP domain that suggests involvement in Rho GTPase signaling pathways, which are crucial for cell migration, proliferation, and cytoskeletal organization . The protein plays key roles in cell proliferation regulation, particularly in lung epithelial repair after injury and tumor progression .

What are the known isoforms of FAM13A and how do they differ functionally?

Research has identified multiple isoforms of FAM13A with distinct functional properties. Two primary isoforms have been characterized in detail: the full-length FAM13A and the shorter FAM13A RhoGAP isoform . The RhoGAP isoform is particularly associated with Th1 gene expression and lung tumor cell migration patterns, while the full-length FAM13A appears more involved in cellular proliferation regulation . These isoforms show differential expression patterns in response to stimuli like TGFβ treatment, with the RhoGAP isoform specifically upregulated during processes involving cell migration . When designing experiments, researchers should consider which isoform they intend to target, as their functional significance varies across different biological contexts.

What are the optimal fixation protocols for immunohistochemistry when using FAM13A antibodies?

For optimal immunohistochemistry (IHC) results with FAM13A antibodies, acetone fixation at -20°C has been successfully employed in studies examining lung adenocarcinoma cells . After fixation, blocking endogenous enzyme activity using dual endogenous enzyme block (e.g., Dako K4065) for approximately 10 minutes is recommended before antibody application . Primary antibody dilutions of 1:1000 have shown effective results with antibodies such as Sigma-Aldrich HPA038109, with an incubation period of 2 hours at room temperature . For developing the signal, labeled Polymer-HPR systems followed by DAB chromogen solution have been effective. Negative controls should always be prepared without primary antibody application while maintaining identical processing for all other steps .

How can researchers effectively detect FAM13A phosphorylation states using antibodies?

Detecting FAM13A phosphorylation requires specialized approaches since the protein undergoes significant post-translational modifications, particularly phosphorylation at serine 312 by AKT kinase . For effective detection, researchers should: 1) Consider using phospho-specific antibodies that target the S312 phosphorylation site, as this modification significantly affects protein stability and function; 2) Include phosphatase inhibitors in all buffer preparations when extracting proteins to prevent dephosphorylation during sample processing; and 3) Compare results with and without AKT inhibitor treatments (e.g., AKT inhibitor VIII at 100 nM) to confirm phosphorylation specificity . Western blotting protocols should be optimized with extended blocking periods (1-2 hours) and overnight primary antibody incubation at 4°C to maximize sensitivity when detecting phosphorylated forms of FAM13A.

What strategies can overcome challenges in detecting endogenous FAM13A protein?

Detecting endogenous FAM13A protein presents challenges due to its regulated expression levels and potential rapid degradation through AKT-mediated phosphorylation and subsequent ubiquitination . Effective strategies include: 1) Pre-treating samples with proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitinated FAM13A; 2) Using cell/tissue-specific extraction protocols optimized for lung tissue or adipocytes where FAM13A is more abundantly expressed ; 3) Employing signal amplification methods such as tyramide signal amplification for IHC applications; and 4) Considering enrichment techniques like immunoprecipitation before Western blotting when working with samples containing low levels of endogenous FAM13A. Additionally, researchers should be aware that FAM13A expression can be rapidly altered in response to stress conditions, so consistent sample handling and prompt processing are essential for reproducible results .

How should experimental protocols be modified when studying FAM13A in different tissue contexts?

FAM13A shows tissue-specific expression patterns and functions, necessitating protocol adjustments based on the tissue being studied. For adipose tissue studies, protocols should account for the upregulation of FAM13A during adipocyte differentiation . When extracting proteins from adipose samples, specialized lysis buffers containing lipid solubilizers improve protein recovery. For lung tissue experiments, researchers should consider the regulatory effects of cigarette smoke extract, which triggers AKT-mediated FAM13A phosphorylation and subsequent degradation . In tumor studies, particularly NSCLC, protocols should account for the relationship between FAM13A and immune regulatory markers (like CTLA4 and HIF1α), potentially requiring co-staining approaches . Each tissue context may require different antibody concentrations, incubation times, and detection methods for optimal results.

What are common sources of false negative results in FAM13A immunoblotting?

False negative results in FAM13A immunoblotting can stem from several factors: 1) Rapid degradation of FAM13A protein via the AKT-triggered CULLIN4A/DCAF1 E3 ligase complex pathway, which necessitates using proteasome inhibitors during sample preparation ; 2) Inefficient protein extraction, particularly from adipose tissues where specialized extraction buffers may be required ; 3) Antibody epitope masking due to post-translational modifications like phosphorylation at serine 312, which may alter antibody recognition ; 4) Inappropriate blocking reagents leading to excessive blocking of epitopes; and 5) Sample-specific protein expression levels that may fall below detection thresholds, especially in models where FAM13A has been knocked down or in tissues with naturally low expression. To address these issues, researchers should consider using positive controls with known FAM13A expression, optimizing protein extraction protocols specifically for their tissue of interest, and potentially using alternative antibodies targeting different epitopes of FAM13A.

How can cross-reactivity issues with FAM13A antibodies be identified and mitigated?

Cross-reactivity concerns with FAM13A antibodies require systematic validation approaches. To identify potential cross-reactivity: 1) Perform parallel experiments using FAM13A knockout or knockdown models alongside wild-type controls to verify antibody specificity ; 2) Conduct peptide competition assays where the antibody is pre-incubated with the immunizing peptide before application; 3) Test the antibody across multiple species if working in comparative models, as FAM13A exhibits sequence conservation but with potential species-specific variations ; and 4) Compare results obtained with multiple antibodies targeting different epitopes of FAM13A. To mitigate identified cross-reactivity, researchers can: 1) Use more stringent washing conditions; 2) Optimize antibody dilutions to minimize non-specific binding; 3) Pre-adsorb antibodies with proteins from relevant knockout tissues; and 4) Consider using monoclonal antibodies with defined epitope specificity for critical experiments where absolute specificity is required.

What are optimal protocols for detecting FAM13A in adipocytes and preadipocytes?

For optimal detection of FAM13A in adipocyte models, researchers should consider the dynamic expression changes during differentiation, as FAM13A expression significantly increases during adipocyte differentiation . For preadipocytes and differentiating adipocytes, effective protocols include: 1) Using differentiation time-course experiments with human cell models such as Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes, with sampling at key differentiation stages ; 2) Employing specialized protein extraction buffers containing mild detergents that effectively solubilize membrane-associated proteins without disrupting antibody epitopes; 3) For immunofluorescence studies, optimizing fixation protocols to preserve lipid droplet structure while maintaining protein antigenicity; and 4) Considering the expression of adipogenesis markers (e.g., CEBPA and PPARG) and beige adipogenic markers (e.g., PGC1A and UCP1) as internal controls to correlate FAM13A expression with differentiation stages . Western blotting protocols may require longer transfer times for larger FAM13A isoforms, particularly in lipid-rich samples.

What considerations are important when using FAM13A antibodies in lung epithelial cells?

When studying FAM13A in lung epithelial cells, researchers should consider several factors that influence detection and interpretation: 1) FAM13A protein levels are dynamically regulated in response to injury, with AKT-mediated downregulation occurring during the acute stress response in lung epithelial cells ; 2) Cigarette smoke extract treatment triggers signaling cascades that lead to FAM13A phosphorylation and subsequent degradation, potentially requiring proteasome inhibitors to preserve protein levels during experimental procedures ; 3) For immunohistochemistry applications in lung tissues, comparison between tumoral and non-tumoral regions requires careful optimization of antibody concentration, as FAM13A expression levels differ between these regions ; 4) When examining lung repair processes, consider co-staining for proliferation markers, as reduced FAM13A levels are associated with accelerated epithelial cell proliferation during recovery phases after injury ; and 5) FAM13A expression patterns in lung epithelial cells should be evaluated in context with immune cell markers and hypoxia-related factors like HIF1α, which show significant correlations with FAM13A expression .

How can FAM13A antibodies be used to study protein-protein interactions?

FAM13A participates in critical protein-protein interactions that influence its function and stability. For studying these interactions, researchers can employ several antibody-dependent approaches: 1) Co-immunoprecipitation (Co-IP) using FAM13A antibodies to pull down interacting partners, particularly focusing on AKT, CULLIN4A/DCAF1 E3 ligase complex components, and Rho GTPase pathway proteins ; 2) Proximity ligation assays (PLA) to visualize and quantify interactions between FAM13A and predicted partners like N-Myc in neuroblastoma or HIF1α in hypoxic conditions ; 3) Chromatin immunoprecipitation (ChIP) followed by sequencing when investigating potential roles of FAM13A in transcriptional regulation complexes; and 4) Reverse phase protein arrays (RPPA) to simultaneously assess multiple signaling pathways potentially influenced by FAM13A. When designing these experiments, researchers should consider that protein interactions may be transient or dependent on specific modifications, such as the phosphorylation of FAM13A at serine 312, which mediates its interaction with the CULLIN4A/DCAF1 E3 ligase complex .

What approaches can verify antibody specificity when studying the structural characteristics of FAM13A?

Verifying antibody specificity is particularly challenging when studying FAM13A structural characteristics, as the experimental structure remains largely unknown and has been primarily predicted through computational approaches like AlphaFold . Researchers should implement multiple validation strategies: 1) Compare antibody recognition patterns against recombinant FAM13A fragments representing different domains, particularly focusing on the RhoGAP domain versus other regions; 2) Perform epitope mapping to precisely identify the recognition site of the antibody, especially important when studying proteins with optimized conformational structures ; 3) Use competitive binding assays with synthetic peptides representing the predicted epitope regions; 4) Validate antibody specificity through genetic approaches by testing recognition in cells with CRISPR-Cas9-mediated FAM13A knockout or targeted domain deletions; and 5) Consider using multiple antibodies targeting different epitopes to build a comprehensive understanding of the protein's structure. For advanced structural studies, researchers might complement antibody approaches with tagged recombinant expression systems that allow structural analysis through techniques like cryo-electron microscopy.

How should researchers interpret contradictory FAM13A antibody staining patterns across different experimental models?

Contradictory FAM13A antibody staining patterns may reflect true biological differences rather than technical artifacts. When encountering such contradictions, researchers should: 1) Systematically evaluate the biological contexts of each model, recognizing that FAM13A functions differ between adipocytes, lung epithelial cells, and cancer cells ; 2) Consider the influence of hypoxic conditions, which significantly affect FAM13A expression and might vary between experimental setups ; 3) Examine the activation state of the AKT pathway, as AKT-mediated phosphorylation triggers FAM13A degradation, potentially leading to reduced staining intensity in models with high AKT activity ; 4) Assess the dominant isoform expression in each model, as antibodies may have different affinities for the full-length FAM13A versus the RhoGAP isoform ; and 5) Evaluate the specific post-translational modification status, which may mask epitopes or alter subcellular localization. To resolve these contradictions, researchers should employ complementary techniques such as RT-qPCR for transcript analysis, fluorescent protein tagging to track real-time localization, and mass spectrometry to identify post-translational modifications specific to each experimental context.

What are best practices for using FAM13A antibodies in tumor microenvironment studies?

For tumor microenvironment studies involving FAM13A, researchers should implement specialized approaches: 1) Use multiplex immunofluorescence to simultaneously detect FAM13A alongside immune cell markers (CD2, CD3D, CD3E, CD8A, TBX21, NKG7) and hypoxia markers (HIF1α), as these show significant correlations with FAM13A expression patterns ; 2) When analyzing spatial distribution, consider the inverse correlation between FAM13A and CTLA4 expression, which has implications for immune checkpoint regulation in tumors ; 3) Employ tissue microarrays containing matched tumor and adjacent normal tissue to standardize staining conditions while assessing tumor-specific expression patterns; 4) Consider dual RNA in situ hybridization with immunohistochemistry to correlate transcript and protein levels, which may diverge due to post-translational regulation of FAM13A; and 5) Integrate single-cell approaches to deconvolute the complex cellular heterogeneity of tumor microenvironments, as FAM13A expression varies across different cell populations . These practices help establish the relationship between FAM13A expression and immune infiltration patterns, which is crucial for understanding its role in cancer progression.

How can FAM13A antibodies be optimized for detecting altered expression in COPD and lung disease models?

Optimizing FAM13A antibody protocols for COPD and lung disease models requires specific considerations: 1) Utilize dual staining approaches that simultaneously detect FAM13A and markers of lung injury or repair, as FAM13A levels fluctuate during these processes ; 2) Implement quantitative analysis methods like tissue cytometry to precisely measure expression changes across different lung compartments (airways, alveoli, vessels); 3) Consider the impact of cigarette smoke exposure timing on FAM13A expression, as acute exposure triggers AKT-mediated degradation while chronic effects may differ significantly ; 4) Compare results across multiple COPD severity stages, as FAM13A expression patterns may evolve with disease progression; and 5) Validate antibody performance in both mouse models and human patient samples, as interspecies differences exist despite sequence conservation . For mechanistic studies, researchers should consider parallel assessment of AKT pathway activation and ubiquitination machinery components like CULLIN4A and DCAF1, which regulate FAM13A protein stability in response to cellular stressors common in COPD pathophysiology .

What protocols can detect FAM13A in extracellular vesicles and secretomes?

Investigating FAM13A in extracellular vesicles (EVs) and secretomes requires specialized approaches beyond standard cellular protein detection: 1) Implement sequential ultracentrifugation protocols optimized for isolating different EV populations, followed by Western blotting with highly sensitive FAM13A antibodies; 2) Consider using EV array technologies that employ antibody-based capture of vesicles followed by detection with FAM13A-specific antibodies; 3) Validate findings with orthogonal techniques like mass spectrometry to confirm FAM13A presence and potential post-translational modifications in extracellular compartments; 4) Use size-exclusion chromatography in combination with Western blotting to separate and characterize different vesicle populations potentially carrying FAM13A; and 5) Implement controlled experimental conditions that account for the potential degradation of FAM13A through the AKT-mediated pathway, which may impact its detection in secreted forms . These approaches help determine whether FAM13A functions extend beyond intracellular roles to include intercellular communication, particularly in contexts like adipose tissue remodeling and tumor microenvironments.

How can single-cell analysis be integrated with FAM13A antibody techniques?

Integrating single-cell analysis with FAM13A antibody techniques provides unprecedented resolution of expression patterns in heterogeneous tissues: 1) Employ mass cytometry (CyTOF) with metal-conjugated FAM13A antibodies alongside lineage and functional markers to profile expression across thousands of individual cells simultaneously; 2) Implement imaging mass cytometry to preserve spatial information while achieving single-cell resolution of FAM13A expression in tissue contexts; 3) Combine index-sorted single-cell RNA sequencing with protein verification using FAM13A antibodies on the same cells to correlate transcript and protein levels at individual cell resolution; 4) Utilize proximity extension assays for high-sensitivity protein detection in limited material from single cells; and 5) Consider computational approaches that integrate antibody-based FAM13A protein measurements with transcriptomic data to identify regulatory networks at single-cell resolution . These integrated approaches are particularly valuable in contexts like tumor heterogeneity studies, where FAM13A expression varies across different cell populations and correlates with immune infiltration patterns and checkpoint marker expression .

What considerations are important when developing antibodies against specific FAM13A post-translational modifications?

Developing antibodies against specific FAM13A post-translational modifications requires careful planning: 1) Prioritize the serine 312 phosphorylation site, as this modification by AKT kinase critically regulates protein stability and degradation ; 2) Design immunizing peptides that precisely mimic the modified region, ensuring appropriate flanking sequences to maintain structural context; 3) Implement rigorous validation protocols including phosphatase treatments, site-directed mutagenesis (S312A mutants), and parallel detection in AKT inhibitor-treated samples ; 4) Consider potential neighboring modifications that might influence epitope recognition, as FAM13A likely undergoes multiple modifications simultaneously; and 5) Validate antibody specificity across different experimental models, as modification patterns may vary between tissue contexts. For advanced applications, researchers should develop quantitative assays that can measure the ratio of modified to unmodified FAM13A, providing deeper insights into the dynamic regulation of this protein in response to stimuli like cigarette smoke extract, hypoxia, or growth factor signaling .