ABCA9 Antibody

What is ABCA9 and why is it significant in research?

ABCA9 (ATP-binding cassette, sub-family A member 9) is a member of the ABC transporter superfamily, specifically the ABC1 subfamily, which is exclusive to multicellular eukaryotes. The protein is ubiquitously expressed, with notably high expression in heart, brain, and fetal tissues. ABCA9 contains two ABC transporter domains and exists in five alternatively spliced isoforms. Its significance stems from its role in monocyte differentiation and lipid homeostasis, being upregulated during monocyte differentiation into macrophages and downregulated by cholesterol loading of macrophages. The gene spans 85 kb on chromosome 17q24, containing 39 exons with a non-coding first exon .

What research applications are validated for ABCA9 antibodies?

Commercial ABCA9 antibodies have been validated for several research applications. These include Western blotting (recommended dilutions ranging from 1:500-1:2000 or 0.04-0.4 μg/mL depending on the specific antibody), immunohistochemistry (dilutions typically 1:2500-1:5000), and ELISA. Some antibodies have also been tested in peptide ELISA with detection limits at dilutions up to 1:128,000. The specific application validations vary by manufacturer, with some antibodies being validated for multiple techniques while others are optimized for particular applications .

How should ABCA9 antibodies be stored and handled for optimal results?

ABCA9 antibodies should generally be stored at -20°C for long-term stability. Many commercial preparations come in a buffered aqueous glycerol solution or with additives like 0.02% sodium azide and 50% glycerol at pH 7.3 to maintain stability. Some lyophilized antibodies require reconstitution in 100 μL of sterile water followed by centrifugation to remove any insoluble material. For storage after reconstitution, dividing into small aliquots is recommended for antibodies without stabilizers like BSA, though some preparations containing 0.1% BSA can be stored without aliquoting. Repeated freeze-thaw cycles should be avoided to maintain antibody integrity and performance .

What are the common cellular and tissue expression patterns of ABCA9?

ABCA9 is ubiquitously expressed in human tissues, with particularly high expression observed in heart, brain, and fetal tissues. At the cellular level, ABCA9 demonstrates membrane subcellular localization consistent with its function as a transporter. Expression is notably dynamic in monocyte-derived cells, with upregulation occurring during monocyte differentiation into macrophages. Conversely, cholesterol loading of macrophages leads to downregulation of ABCA9. This expression pattern aligns with its proposed role in lipid transport and homeostasis. The Human Protein Atlas project has conducted extensive tissue microarray analysis across 44 normal human tissues and 20 common cancer types, providing detailed information on ABCA9 distribution patterns .

How should positive and negative controls be selected for ABCA9 antibody validation?

For effective ABCA9 antibody validation, positive controls should include tissues or cell lines with known expression of ABCA9. Based on expression data, heart and brain tissue samples or cells derived from these tissues would serve as appropriate positive controls. U-251 cells have been specifically validated as a positive control for Western blot applications. For negative controls, researchers should consider using tissues where ABCA9 expression is minimal or knockout/knockdown models where available. Additionally, peptide blocking experiments using the immunogen peptide (such as FWFQHGRANHVVLENETDSDPTPNDCFEP for some commercially available antibodies) can provide specificity confirmation. Some manufacturers offer antigen controls specifically designed for their antibodies, which can be found in their product linkage sections .

What sample preparation protocols yield optimal results for ABCA9 detection?

For optimal ABCA9 detection across different applications, specific sample preparation protocols are recommended:

Western Blot Sample Preparation:

• Extract proteins using a lysis buffer containing protease inhibitors

• For membrane proteins like ABCA9, consider detergent-based extraction methods

• Heat samples at 95°C for 5 minutes in reducing sample buffer

• Load 20-50 μg of protein per lane

Immunohistochemistry Sample Preparation:

• Fix tissues in 10% neutral buffered formalin

• Perform antigen retrieval (method may vary by antibody)

• Block endogenous peroxidase and non-specific binding

• Apply antibody at recommended dilution (typically 1:2500-1:5000)

The observed molecular weight for ABCA9 detection is reported at 31 kDa for some antibodies, though this may represent a specific fragment or isoform of the full-length protein .

How can cross-reactivity issues be identified and mitigated with ABCA9 antibodies?

Identifying and mitigating cross-reactivity with ABCA9 antibodies requires a multi-faceted approach:

Identification Methods:

• Examine reactivity against protein arrays (some manufacturers test against 364 human recombinant protein fragments)

• Perform Western blot analysis across multiple cell lines with varying ABCA9 expression

• Compare results from multiple antibodies targeting different epitopes

• Include genetic models (knockdown/knockout) as definitive controls

Mitigation Strategies:

• Select antibodies generated against unique epitopes (like FWFQHGRANHVVLENETDSDPTPNDCFEP) that are not conserved in related proteins

• Use affinity-purified antibodies, which generally have lower cross-reactivity

• Optimize blocking conditions and antibody dilutions

• Consider pre-absorption with related proteins when working in complex samples

Particularly important is evaluating potential cross-reactivity with other ABCA family members, as the ABC1 subfamily contains multiple related proteins clustered on chromosome 17q24 .

What techniques are available for studying ABCA9's role in lipid homeostasis?

To investigate ABCA9's role in lipid homeostasis, researchers can employ several sophisticated techniques:

Functional Transport Assays:

• Radiolabeled lipid efflux assays measuring transport activity

• Fluorescently labeled lipid tracking in live cells expressing ABCA9

• Lipidomic analysis of cells with modulated ABCA9 expression

Regulation Studies:

• Cholesterol loading experiments, which have been shown to downregulate ABCA9

• Time-course analysis during monocyte-to-macrophage differentiation

• Reporter assays to identify transcriptional regulators

Interaction Studies:

• Co-immunoprecipitation to identify protein binding partners

• Proximity ligation assays for in situ interaction detection

• FRET/BRET approaches for dynamic interaction monitoring

These methodologies can elucidate how ABCA9 contributes to cellular lipid homeostasis and how its activity is regulated in response to changing lipid environments .

How can ABCA9 antibodies be used to study protein-protein interactions?

ABCA9 antibodies can be instrumental in studying protein-protein interactions through several methodologies:

Co-immunoprecipitation (Co-IP):

• Use anti-ABCA9 antibodies (like PA5-18953 or HPA054823) conjugated to beads

• Precipitate ABCA9 from cell lysates under non-denaturing conditions

• Identify co-precipitating proteins via mass spectrometry or Western blot

Proximity-based Methods:

• Proximity Ligation Assay (PLA) using anti-ABCA9 paired with antibodies against potential interactors

• BioID or APEX proximity labeling with ABCA9 as the bait protein

• FRET/BRET approaches using fluorescent protein-tagged ABCA9

Pull-down Assays:

• Express recombinant ABCA9 domains (particularly the ATP-binding cassette domains)

• Use purified domains as bait in pull-down experiments

• Validate interactions with antibody detection

When selecting antibodies for these applications, researchers should choose those validated for immunoprecipitation or that recognize native (non-denatured) ABCA9 .

What methodologies are recommended for studying ABCA9 in macrophage differentiation?

For studying ABCA9 in macrophage differentiation, a multi-dimensional approach is recommended:

Expression Analysis During Differentiation:

• Time-course Western blot analysis using validated ABCA9 antibodies (1:500-1:2000 dilution)

• qRT-PCR for transcript level monitoring

• Single-cell analysis to identify heterogeneity in expression

Functional Impact Assessment:

• siRNA/shRNA knockdown or CRISPR-Cas9 knockout of ABCA9

• Phenotypic characterization of differentiation markers

• Functional assessment of macrophage activities (phagocytosis, cytokine production)

Regulatory Mechanism Exploration:

• ChIP-seq to identify transcription factors binding to ABCA9 promoter during differentiation

• Promoter-reporter assays to validate regulatory elements

• DNA methylation and histone modification analysis

Experimental Design Table for Macrophage Differentiation Studies:

This systematic approach enables comprehensive characterization of ABCA9's role in the differentiation process .

What are common technical challenges when detecting ABCA9 and how can they be overcome?

Researchers frequently encounter several technical challenges when detecting ABCA9:

Low Signal Intensity:

• Challenge: ABCA9 may be expressed at low levels in some cell types.

• Solution: Use signal amplification methods such as TSA (Tyramide Signal Amplification), more sensitive detection systems, or longer exposure times. Consider concentrating protein samples through immunoprecipitation before Western blotting.

Multiple Bands in Western Blots:

• Challenge: Detection of multiple bands can complicate interpretation.

• Solution: Verify the expected molecular weight (reported as 31 kDa for some antibodies, though the full protein is larger). Consider that ABCA9 exists in five alternatively spliced isoforms, which may appear as different bands. Use positive controls with known expression patterns and peptide competition assays to confirm specificity.

Background Staining in IHC/ICC:

• Challenge: High background can mask specific signals.

• Solution: Optimize blocking conditions (BSA, serum, commercial blockers), increase washing times and volumes, and titrate the primary antibody. For some applications, dilutions of 1:2500-1:5000 are recommended for optimal signal-to-noise ratio.

Sample Preparation Issues:

• Challenge: ABCA9 is a membrane protein, which can be difficult to extract.

• Solution: Use specialized membrane protein extraction buffers containing appropriate detergents. For fixed tissues, optimize antigen retrieval methods (heat-induced vs. enzymatic) to expose epitopes without damaging tissue morphology .

How should researchers interpret contradictory results between different detection methods?

When faced with contradictory results between different detection methods for ABCA9:

Systematic Analysis Approach:

• Evaluate method-specific limitations: Western blot detects denatured proteins, while IHC may detect native conformations. ELISA might capture soluble fragments. These inherent differences can explain some contradictions.

• Antibody epitope considerations: Different antibodies (like PA5-18953 vs. HPA054823) target different epitopes that may be differentially accessible in various applications. Cross-reference the immunogen sequence information.

• Isoform-specific detection: ABCA9's five alternatively spliced isoforms may be differentially detected by different methods or antibodies.

• Protocol optimization verification: Ensure each method has been properly optimized with appropriate controls.

Resolution Strategies:

• Use multiple antibodies targeting different epitopes and compare results

• Complement protein-level detection with mRNA analysis (qPCR, RNA-seq)

• Employ genetic approaches (siRNA knockdown, CRISPR knockout) to confirm specificity

• Consider functional assays that don't rely solely on antibody detection

The integration of multiple methods provides a more robust understanding of ABCA9 expression and function than relying on a single technique .

What criteria should be used to evaluate ABCA9 antibody specificity in publications?

When evaluating ABCA9 antibody specificity in publications, researchers should apply these critical assessment criteria:

Fundamental Validation Evidence:

• Genetic controls: Use of ABCA9 knockout/knockdown models as negative controls

• Peptide competition: Demonstration that the immunizing peptide blocks antibody binding

• Orthogonal detection methods: Correlation between antibody-based detection and mRNA levels

• Expected expression pattern: Consistency with known ABCA9 expression (high in heart, brain, and fetal tissues)

Technical Validation Parameters:

• Complete antibody identification: Catalog number, lot number, dilution used, and vendor

• Control documentation: Clear description and presentation of positive and negative controls

• Expected molecular weight: Confirmation of detection at appropriate size (full-length or specific fragments)

• Cross-reactivity assessment: Testing against related proteins, particularly other ABCA family members

Reproducibility Indicators:

• Multiple antibodies: Consistent results with antibodies targeting different epitopes

• Independent replications: Similar findings across different experiments or research groups

• Quantification methods: Objective measurement approaches rather than representative images alone

Publications should ideally include these validation elements to establish confidence in the specificity of ABCA9 antibody-based findings .

How might ABCA9 antibodies contribute to understanding disease mechanisms?

ABCA9 antibodies offer significant potential for elucidating disease mechanisms across several domains:

Cardiovascular Diseases:
Given ABCA9's high expression in heart tissue and its role in lipid homeostasis, antibodies can help investigate its contribution to atherosclerosis, heart failure, and cardiomyopathies. Immunohistochemical analysis of diseased versus healthy heart tissues could reveal alterations in expression patterns or subcellular localization.

Neurodegenerative Disorders:
With substantial expression in brain tissue, ABCA9 may play roles in lipid metabolism disorders affecting neuronal function. Antibodies can help map expression in specific brain regions and neural cell types, potentially revealing associations with conditions like Alzheimer's disease or other disorders involving lipid dysregulation.

Cancer Biology:
The Human Protein Atlas has examined ABCA9 expression across 20 common cancer types. Further antibody-based studies could reveal whether expression changes correlate with cancer progression, metastasis, or treatment response, potentially identifying ABCA9 as a biomarker or therapeutic target.

Immunological Disorders:
Given ABCA9's dynamic regulation during monocyte-to-macrophage differentiation, antibodies can help investigate whether dysregulated expression contributes to inflammatory diseases, immune deficiencies, or autoimmune conditions .

What novel techniques are emerging for studying ABCA9 protein dynamics?

Emerging techniques for studying ABCA9 protein dynamics represent cutting-edge approaches in molecular biology:

Live-Cell Imaging Approaches:

• CRISPR-mediated endogenous tagging: Inserting fluorescent tags at the endogenous ABCA9 locus to monitor native protein dynamics

• Photo-switchable fluorescent protein fusions: Enabling pulse-chase experiments to track ABCA9 movement and turnover

• Lattice light-sheet microscopy: Providing high-resolution, low-phototoxicity imaging of ABCA9 trafficking

Structural Biology Advancements:

• Cryo-EM analysis: Revealing the 3D structure of ABCA9 in different conformational states

• Hydrogen-deuterium exchange mass spectrometry: Probing dynamic regions and binding interfaces

• Single-molecule FRET: Measuring conformational changes during transport cycles

Systems Biology Integration:

• Spatial proteomics: Mapping ABCA9's precise subcellular localization and co-localization networks

• Proximity labeling proteomics (BioID, APEX): Identifying context-specific interaction partners

• Multi-omics integration: Correlating ABCA9 dynamics with transcriptomic and metabolomic changes

These emerging techniques complement traditional antibody-based approaches and provide unprecedented insights into ABCA9 function at molecular and cellular levels .

How can researchers contribute to improving ABCA9 antibody validation standards?

Researchers can significantly advance ABCA9 antibody validation standards through several strategic approaches:

Implementation of Comprehensive Validation Protocols:

• Systematically validate antibodies across multiple techniques (WB, IHC, IP, ELISA, ICC)

• Test across diverse cell types and tissues with varying expression levels

• Document and publish complete validation data, even negative results

Development of Reference Materials:

• Create and share ABCA9 knockout cell lines as definitive negative controls

• Generate overexpression systems with tagged ABCA9 as positive controls

• Produce recombinant ABCA9 fragments representing different domains and isoforms

Methodological Standardization:

• Establish optimized protocols for sample preparation specific to ABCA9 detection

• Define consensus criteria for evaluating antibody specificity and sensitivity

• Develop quantitative metrics for antibody performance comparison

Community Engagement and Data Sharing:

• Contribute detailed antibody validation data to repositories like Antibodypedia

• Participate in multi-laboratory validation studies

• Report detailed antibody information in publications (catalog number, lot, dilution, validation)

By adopting these practices, researchers can collectively improve confidence in ABCA9-related findings and accelerate progress in understanding this important transporter protein .

What are the recommended best practices for ABCA9 antibody selection and validation?

For optimal outcomes in ABCA9 research, following these best practices for antibody selection and validation is crucial:

Selection Criteria:

• Choose antibodies with comprehensive validation data across multiple techniques

• Consider the specific epitope targeted and its conservation across species or isoforms

• Select antibodies validated in applications matching your experimental needs

• When possible, use antibodies that have been validated by independent sources

Validation Workflow:

• Begin with positive controls (U-251 cells have been validated for Western blot)

• Include negative controls (ideally ABCA9 knockdown/knockout samples)

• Perform peptide competition assays to confirm specificity

• Validate across multiple applications if the antibody will be used in different techniques

Application-Specific Considerations:

• For Western blot: Optimize protein extraction for membrane proteins, use recommended dilutions (1:500-1:2000)

• For IHC/ICC: Carefully optimize antigen retrieval and antibody dilution (1:2500-1:5000 recommended)

• For IP: Verify antibody capacity to recognize native (non-denatured) ABCA9

Documentation Standards:

• Maintain detailed records of antibody source, lot number, and validation experiments

• Include complete methods sections in publications with all relevant antibody information

• Share validation data with the research community to advance collective knowledge

Following these practices ensures reliable, reproducible research findings related to ABCA9 .

How can researchers integrate antibody-based detection with other techniques for comprehensive ABCA9 characterization?

A multi-modal approach to ABCA9 characterization yields the most comprehensive understanding:

Complementary Technique Integration Strategy:

• Protein-Level Characterization:

  • Antibody-based detection (Western blot, IHC, ICC) for protein expression and localization

  • Mass spectrometry for unbiased protein identification and post-translational modifications

  • Proximity labeling (BioID, APEX) for interaction network mapping

• Genetic-Level Correlation:

  • RT-qPCR and RNA-seq to correlate protein expression with transcript levels

  • CRISPR-Cas9 modification for functional studies and antibody validation

  • ChIP-seq to identify transcriptional regulators of ABCA9 expression

• Functional Characterization:

  • Transport assays to assess ABCA9's role in lipid movement

  • Live-cell imaging with fluorescent lipid probes

  • Lipidomics to identify specific lipid species affected by ABCA9 activity

Integration Workflow Example:

This integrated approach provides validation through methodological triangulation while yielding deeper insights than any single technique alone .

What key considerations should guide experimental design for ABCA9 studies?

Effective ABCA9 studies require careful experimental design considerations:

Biological Context Optimization:

• Cell type selection: Choose models with physiologically relevant ABCA9 expression (cardiac cells, brain-derived cells, macrophages) or compare across a panel of cell types.

• Differentiation state: For monocyte/macrophage studies, carefully control and document differentiation stages, as ABCA9 expression changes dramatically during this process.

• Lipid environment: Consider how experimental conditions affect cellular lipid status, which can regulate ABCA9 expression (cholesterol loading downregulates ABCA9).

Technical Parameter Optimization:

• Antibody selection: Choose antibodies validated for your specific application; different antibodies may be optimal for Western blot (1:500-1:2000) versus IHC (1:2500-1:5000).

• Sample preparation: For membrane proteins like ABCA9, extraction methods significantly impact detection success.

• Controls design: Include both positive controls (tissues/cells with known expression) and negative controls (knockdown/knockout samples where possible).

Experimental Design Framework:

• Hypothesis-driven approach: Clearly define how ABCA9 may function in your system of interest.

• Power analysis: Ensure sufficient replication to detect biologically meaningful changes.

• Temporal considerations: Plan time-course experiments when studying dynamic processes like differentiation.

• Multi-level validation: Confirm key findings using complementary techniques or different antibodies.