ABCA3 Antibody

What is ABCA3 and where is it primarily expressed?

ABCA3 (ATP-binding cassette subfamily A member 3) belongs to the ATP-binding cassette transporter superfamily. It is predominantly expressed in alveolar type II pneumocytes where it localizes to the limiting membrane of lamellar bodies . ABCA3 plays a critical role in pulmonary surfactant synthesis, likely through the transportation of lipids such as cholesterol . While ABCA3 mRNA has been detected across multiple tissues including lung, liver, kidney, cerebellum, and gastrointestinal tract, protein expression at detectable levels by immunohistochemistry is primarily observed in lung tissue and Purkinje cells of the cerebellum . During development, ABCA3 expression in fetal mouse lung increases progressively from relatively low levels at embryonic day 14 (E14) to higher levels prior to birth .

What applications are most reliable for ABCA3 antibody usage?

Based on validation data from multiple antibody suppliers, Western blot (WB) is the most consistently reliable application for ABCA3 antibodies, with recommended dilutions typically ranging from 1:500 to 1:2000 . Immunohistochemistry (IHC) and immunofluorescence (IF) are also well-established applications, particularly for lung tissue samples . Additionally, some ABCA3 antibodies have been validated for ELISA, immunocytochemistry (ICC), and electron microscopy (EM), especially when studying localization within lamellar bodies . When selecting an antibody, researchers should evaluate the specific validation data for each application and choose products with demonstrated reactivity to their species of interest (commonly human, mouse, or rat) .

How should sample preparation be optimized for ABCA3 detection?

For optimal ABCA3 detection, sample preparation is critical. In Western blot applications, unboiled rat lung tissue samples have been specifically noted to yield positive results . This suggests that ABCA3's structural integrity may be heat-sensitive, and native protein conditions might be preferable for certain applications. For immunohistochemistry in tissues outside the lung, antigen retrieval methods may be necessary, as demonstrated by the detection of ABCA3 in mouse cerebellum Purkinje cells requiring antigen retrieval when using certain antibodies . When working with cultured cells, such as type II alveolar cells or cell lines like MLE-13 and MLE-15, standard fixation with paraformaldehyde followed by permeabilization is generally effective for immunofluorescence applications .

What positive controls are recommended for ABCA3 antibody validation?

For ABCA3 antibody validation, rat lung tissue serves as an excellent positive control for Western blot applications . The observed molecular weight of ABCA3 protein is typically 140-160 kDa . Rat kidney extracts have also been successfully used as positive controls, as demonstrated in Western blot analyses with 1:1000 antibody dilution . For cellular studies, immortalized mouse lung epithelial cell lines (MLE-13 and MLE-15) and purified type II epithelial cells isolated from adult mouse lung have shown detectable ABCA3 expression . Human cell lines such as HeLa cells can also serve as positive controls for immunofluorescence applications, although the expression level may vary . Researchers should note that despite detection of ABCA3 mRNA in multiple tissues, protein expression at detectable levels is more restricted, with consistent results in lung tissue and cerebellum .

How can researchers quantify functional impairment of ABCA3 variants?

Quantifying functional impairment of ABCA3 variants requires a multi-parameter approach that systematically captures data from different assays. Researchers should implement the following methodology:

• Establish quantitative cellular readouts that can be compared between different studies, rather than using qualitative assessments .

• Incorporate PC (phosphatidylcholine) recycling assays, which have demonstrated benefit over de novo PC synthesis as a functional variable for assessing ABCA3 activity .

• Combine multiple functional aspects in the assessment, including:

  • Intracellular trafficking analysis

  • Lipid transport capacity measurement

  • Protein stability evaluation

  • Cellular localization studies

• Correlate in vitro results with clinical outcomes when possible to validate the biological significance of observed impairments .

It is important to note that in silico predictions and structure analysis alone are currently insufficient to reliably predict the effects of ABCA3 variants, as interpretation based solely on ACMG guidelines has failed to consistently correlate with clinical outcomes . Therefore, comprehensive functional testing remains essential for accurate variant characterization.

What technical considerations are important when using ABCA3 antibodies for different detection methods?

When using ABCA3 antibodies across different detection methods, several technical considerations must be addressed:

For Western Blot:

• Sample preparation is critical - unboiled rat lung tissue samples have been specifically noted to yield positive results, suggesting heat sensitivity of the ABCA3 protein .

• Use the appropriate dilution range (typically 1:500-1:2000) and titrate the antibody in each testing system for optimal results .

• The observed molecular weight should be 140-160 kDa; significant deviations may indicate degradation or non-specific binding .

For Immunohistochemistry/Immunofluorescence:

• Antigen retrieval methods may be necessary for certain tissues, as demonstrated by the detection of ABCA3 in mouse cerebellum requiring antigen retrieval with specific antibodies .

• Different antibodies show variable reactivity in tissues outside the lung, likely dependent on expression levels, accessibility of the ABCA3 antigenic epitopes, and antibody affinity .

• Blocking with recombinant ABCA3 peptide (5-200 μg/ml) can serve as an important specificity control .

For Electron Microscopy:

• Only select antibody clones (such as clone 4D0) have been validated for EM applications, making antibody selection particularly critical for this technique .

• Special fixation and embedding protocols may be required to preserve antigenicity while maintaining ultrastructural details of lamellar bodies.

How do expression patterns of ABCA3 during development inform experimental design?

ABCA3 expression during development follows a specific pattern that should inform experimental design choices:

In fetal mouse lung, ABCA3 mRNA is present at relatively low levels at embryonic day 14 (E14), with expression increasing progressively prior to birth . This developmental pattern coincides with surfactant production in preparation for the transition to air breathing. When designing developmental studies, researchers should consider:

• Temporal sampling strategy: Include multiple developmental timepoints (at least E14, E16, E18, and postnatal) to capture the dynamic expression changes.

• Technical sensitivity requirements: Detection methods for early developmental stages require higher sensitivity due to lower expression levels.

• Comparative analysis approach: Always include adult lung samples as positive controls to establish baseline expression levels.

• Methodological combinations: Combine mRNA quantification (RT-PCR) with protein detection methods to distinguish between transcriptional and post-transcriptional regulation .

• Species considerations: While developmental patterns are generally conserved between mice and humans, the exact timing differs, necessitating species-specific validation.

This developmental expression pattern has significant implications for studies investigating congenital surfactant deficiencies, as mutations affecting ABCA3 expression or function during critical developmental windows may have particularly severe consequences .

What experimental approaches can resolve contradictory results between different ABCA3 antibody clones?

When faced with contradictory results using different ABCA3 antibody clones, researchers should implement the following systematic approach to resolve discrepancies:

• Epitope mapping comparison:

  • Review the immunogen information for each antibody to determine if they target different regions of the ABCA3 protein .

  • Monoclonal antibodies (such as clone 4D0) recognize single epitopes, while polyclonal antibodies recognize multiple epitopes, which may explain differential reactivity .

• Validation with knockout/knockdown controls:

  • Employ ABCA3 knockout or knockdown models as negative controls to definitively assess antibody specificity.

  • siRNA-mediated knockdown in cell lines can serve as temporary alternatives when knockout models are unavailable.

• Peptide competition assay:

  • Perform blocking experiments by preincubating antibodies with increasing concentrations (5-200 μg/ml) of recombinant ABCA3 peptide to confirm specificity .

  • Complete signal abolishment indicates true specificity, while partial reduction may suggest cross-reactivity.

• Multi-method confirmation:

  • Employ orthogonal detection methods (e.g., if discrepancies appear in IHC, confirm with Western blot or RT-PCR) .

  • Correlation between protein detection and mRNA expression can help distinguish true signal from artifact.

• Tissue/sample-dependent optimization:

  • Different antibodies may perform optimally in different sample types - compare performance in fresh versus fixed tissues, or native versus denatured proteins .

  • Systematic testing of various sample preparation methods can identify protocol-dependent reactivity issues.

By implementing this comprehensive approach, researchers can identify whether discrepancies stem from technical issues, epitope accessibility differences, or actual biological variations in ABCA3 expression or modification states.

What are the optimal storage conditions for preserving ABCA3 antibody activity?

For maximum stability and longevity of ABCA3 antibodies, implement the following storage protocol:

Some ABCA3 antibody preparations (particularly those at 20μl sizes) contain 0.1% BSA as a stabilizer . When working with these preparations, it's important to consider potential interactions with assay systems that might be sensitive to BSA. For routine laboratory use, antibodies can be temporarily stored at 4°C for up to one month during active experimental periods, but long-term storage should always revert to -20°C to prevent degradation and loss of activity.

How can researchers optimize Western blot protocols specifically for ABCA3 detection?

To achieve optimal ABCA3 detection in Western blot applications, researchers should implement this specialized protocol:

• Sample preparation:

  • Use unboiled rat lung tissue for positive controls, as heat treatment may disrupt ABCA3 epitopes .

  • For cell lysates, use gentle lysis buffers containing protease inhibitors to prevent degradation of this large protein (140-160 kDa).

• Gel electrophoresis:

  • Employ lower percentage (6-8%) SDS-PAGE gels to allow proper resolution of the 140-160 kDa ABCA3 protein.

  • Load adequate protein (25μg per lane recommended) to ensure detection of potentially low-abundance ABCA3 .

• Transfer conditions:

  • Use wet transfer methods with longer transfer times (overnight at low voltage) to ensure complete transfer of this high molecular weight protein.

  • Consider adding SDS (0.1%) to the transfer buffer to facilitate migration of large proteins.

• Antibody incubation:

  • Titrate primary antibody concentration within the recommended range (1:500-1:2000) .

  • For secondary antibody, a 1:10000 dilution of HRP Goat Anti-Rabbit IgG (H+L) has been validated .

• Detection system:

  • Use enhanced chemiluminescence (ECL) detection with exposure times around 90 seconds as a starting point .

  • For weaker signals, consider using more sensitive detection reagents or longer exposure times.

• Blocking:

  • 3% nonfat dry milk in TBST has been successfully used as blocking buffer .

Following this optimized protocol will maximize the likelihood of specific and sensitive detection of ABCA3 protein in Western blot applications.

What strategies can be used to improve ABCA3 antibody specificity in immunohistochemistry?

To enhance ABCA3 antibody specificity in immunohistochemistry applications, implement the following comprehensive strategy:

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods, as certain ABCA3 epitopes (particularly in tissues like cerebellum) require specific retrieval conditions .

  • Compare heat-induced epitope retrieval using citrate buffer (pH 6.0) versus Tris-EDTA buffer (pH 9.0) to determine optimal conditions for your specific tissue and antibody.

• Antibody validation controls:

  • Include peptide competition controls by preincubating the antibody with increasing concentrations (5-200 μg/ml) of recombinant ABCA3 peptide to confirm specificity .

  • Always include known positive tissue controls (lung) alongside experimental samples for comparison.

• Signal amplification considerations:

  • For tissues with lower ABCA3 expression, employ tyramide signal amplification or polymer-based detection systems to enhance sensitivity without increasing background.

  • When using amplification methods, include additional negative controls (omitting primary antibody) to assess potential non-specific signal.

• Tissue-specific protocol adjustments:

  • Despite ABCA3 mRNA detection in multiple tissues, protein detection requires tissue-specific protocol adjustments .

  • Different antibodies show variable reactivity patterns in tissues outside the lung, necessitating antibody selection based on the specific tissue being studied .

• Multi-antibody verification:

  • For critical experiments, verify results using multiple antibodies targeting different ABCA3 epitopes.

  • Correlation of staining patterns between different antibodies significantly increases confidence in specificity.

Implementation of these strategies will allow researchers to differentiate between true ABCA3 expression and non-specific background, particularly in tissues where ABCA3 is expressed at lower levels compared to lung tissue.

How can ABCA3 antibodies be utilized to study the pathophysiology of surfactant deficiency disorders?

ABCA3 antibodies serve as crucial tools in investigating surfactant deficiency disorders through several methodological approaches:

• Mutation-specific trafficking analysis:

  • Utilize immunofluorescence with ABCA3 antibodies to assess intracellular localization patterns of mutant ABCA3 proteins in patient-derived samples or transfected cell models .

  • Compare co-localization with organelle markers to determine if specific mutations cause trafficking defects versus functional defects of properly localized protein.

• Quantitative expression analysis:

  • Implement Western blot analysis with standardized quantification to compare ABCA3 protein levels between patient samples and controls .

  • Correlate protein levels with mRNA expression to differentiate between transcriptional regulation issues and post-translational processing defects.

• Functional correlation studies:

  • Combine ABCA3 antibody-based detection with phosphatidylcholine (PC) recycling assays to establish structure-function relationships of specific mutations .

  • This integrated approach has demonstrated superior sensitivity compared to de novo PC synthesis assays in detecting functional impairments.

• Diagnostic algorithm development:

  • Incorporate antibody-based detection methods into systematic approaches for variant interpretation that move beyond in silico predictions .

  • Correlate cellular phenotypes detected by antibodies with clinical outcomes to develop more accurate predictive models.

This multifaceted approach using ABCA3 antibodies enables researchers to mechanistically classify surfactant deficiency disorders, potentially leading to tailored therapeutic strategies based on the specific molecular defect.

What technical considerations should researchers address when studying ABCA3 in non-pulmonary tissues?

When investigating ABCA3 in non-pulmonary tissues, researchers must address several critical technical considerations:

• Detection sensitivity requirements:

  • ABCA3 expression levels in non-pulmonary tissues are significantly lower than in lung tissue, necessitating more sensitive detection methods .

  • While ABCA3 mRNA has been detected in liver, kidney, cerebellum, and gastrointestinal tract by RT-PCR, protein detection by standard immunohistochemistry has been consistently successful only in cerebellum (Purkinje cells) .

• Antibody selection strategy:

  • Different ABCA3 antibodies demonstrate variable reactivity in non-pulmonary tissues, likely due to differences in epitope accessibility and antibody affinity .

  • Systematic testing of multiple antibodies (both monoclonal and polyclonal) is recommended for each non-pulmonary tissue of interest.

• Protocol modifications for specific tissues:

  • For cerebellar detection, antigen retrieval methods may be required with certain antibodies (e.g., GP985) .

  • Despite detecting ABCA3 mRNA in various tissues, standard IHC protocols have failed to detect ABCA3 protein in adult mouse liver, pancreas, salivary glands, digestive tract, kidney, adrenal, spleen, heart, thymus, thyroid, trachea, most brain regions, and reproductive organs .

• Multi-method verification approach:

  • Combine protein detection (IHC/IF/WB) with mRNA detection methods (RT-PCR) to distinguish between absence of expression versus technical limitations in detection .

  • Consider more sensitive methods such as proximity ligation assay or immunoprecipitation followed by mass spectrometry for tissues with very low expression levels.

By addressing these technical considerations, researchers can avoid false negative results and accurately characterize the true expression pattern of ABCA3 across different tissue types.

How can researchers design experiments to study ABCA3 interactions with other proteins and lipids?

To effectively investigate ABCA3 interactions with other proteins and lipids, researchers should design experiments incorporating these methodological approaches:

• Co-immunoprecipitation studies:

  • Use validated ABCA3 antibodies for immunoprecipitation followed by mass spectrometry to identify interacting protein partners .

  • Confirm findings through reverse co-immunoprecipitation using antibodies against identified partner proteins.

  • Important controls include non-specific IgG precipitations and validation in multiple cell types (primary cells and cell lines).

• Proximity-based interaction assays:

  • Implement proximity ligation assays (PLA) to detect ABCA3 interactions with suspected partner proteins in situ.

  • This approach is particularly valuable for detecting transient interactions that may be disrupted during traditional co-immunoprecipitation.

• Lipid transport assessment:

  • Combine ABCA3 antibody-based detection with PC recycling assays, which have demonstrated superior sensitivity compared to de novo PC synthesis in detecting ABCA3 functional capacity .

  • Implement lipidomic analysis of isolated lamellar bodies to correlate ABCA3 expression/localization with lipid composition.

• Functional domain mapping:

  • Use epitope-specific antibodies targeting different domains of ABCA3 to determine which regions are essential for specific protein-protein or protein-lipid interactions.

  • Compare wild-type ABCA3 with specific domain mutants to identify interaction interfaces.

• Membrane microdomain analysis:

  • Investigate ABCA3 distribution in membrane microdomains using detergent resistance fractionation followed by immunoblotting.

  • Co-localization studies with lipid raft markers can provide insights into the membrane environment where ABCA3 functions.

Through these comprehensive approaches, researchers can elucidate the complex interaction network of ABCA3, potentially identifying novel therapeutic targets for surfactant-related disorders.

What are common issues when using ABCA3 antibodies and how can they be resolved?

When working with ABCA3 antibodies, researchers frequently encounter these challenges, which can be addressed through specific technical interventions:

Implementation of these targeted troubleshooting strategies will significantly improve the reliability and reproducibility of results when working with ABCA3 antibodies across different experimental applications.

How can researchers address conflicting data between ABCA3 mRNA expression and protein detection?

When confronted with discrepancies between ABCA3 mRNA expression and protein detection, researchers should implement this systematic investigative approach:

• Technical validation:

  • Confirm mRNA detection specificity through sequence verification of PCR products or use of multiple primer sets targeting different exons .

  • Validate protein detection methods using known positive controls (lung tissue) alongside experimental samples.

• Biological explanation assessment:

  • Consider post-transcriptional regulation mechanisms (microRNAs, RNA-binding proteins) that might prevent translation despite mRNA presence.

  • Investigate protein stability and turnover rates, which may result in low steady-state protein levels despite detectable mRNA.

• Detection sensitivity comparisons:

  • Recognize that RT-PCR for mRNA detection typically offers greater sensitivity than antibody-based protein detection methods .

  • In tissues with low expression, standard IHC/IF may fail to detect protein that is present below the method's detection threshold.

• Protocol optimization strategy:

  • For tissues with positive mRNA but negative protein detection, implement more sensitive protein detection methods:

    • Signal amplification systems for IHC/IF

    • Concentrated protein extracts for Western blot

    • Immunoprecipitation followed by Western blot for enrichment

• Method integration approach:

  • Implement in situ hybridization alongside IHC on serial sections to directly compare mRNA and protein localization patterns at the cellular level.

  • This comprehensive approach can identify specific cell populations where post-transcriptional regulation may occur.

Through this systematic approach, researchers can determine whether discrepancies represent true biological regulation or technical limitations, advancing understanding of ABCA3 expression patterns across different tissues and developmental stages.