ACO2 Antibody

What is ACO2 and why are ACO2 antibodies important in research?

ACO2 (Aconitase 2) is a mitochondrial enzyme that catalyzes the isomerization of citrate to isocitrate via cis-aconitate intermediate as part of the Krebs cycle . This 85 kDa protein plays a crucial role in cellular energy metabolism and has been implicated in several neurological disorders .

ACO2 antibodies are important research tools because:

• They enable detection and quantification of ACO2 protein levels in various experimental systems

• They facilitate investigation of mitochondrial function and dysfunction

• They help researchers study the role of ACO2 in neurodegenerative conditions like infantile cerebellar retinal degeneration (ICRD) and optic neuropathies

• They allow for the assessment of mutations' effects on protein expression, localization, and function

The relationship between ACO2 and iron regulatory proteins, particularly IRP1 (which can either bind to iron-responsive elements or possess aconitase activity in its Fe-containing form), makes these antibodies valuable for studying iron metabolism and its regulation .

What applications are ACO2 antibodies suitable for?

ACO2 antibodies have been validated for multiple experimental applications. When selecting an antibody for your research, it's important to ensure it has been specifically validated for your intended application.

Researchers should always perform optimization experiments to determine the optimal antibody concentration for their specific experimental conditions, sample types, and detection methods. Appropriate positive and negative controls should be included to validate antibody specificity .

How do you validate ACO2 antibody specificity?

Validation of antibody specificity is critical for obtaining reliable research results. For ACO2 antibodies, consider these methodological approaches:

• Western blot analysis with multiple cell lines: Compare ACO2 detection across different cell types known to express the protein (e.g., HEK-293T, A431, HeLa, HepG2, and Jurkat cells) . The expected molecular weight for ACO2 is approximately 85 kDa .

• Knockout/knockdown controls: Use ACO2 knockout cells or siRNA-mediated knockdown samples as negative controls to confirm antibody specificity.

• Cross-reactivity testing: If working with non-human samples, verify the species cross-reactivity of your antibody. Many ACO2 antibodies react with human, mouse, rat, and monkey samples, but validation is essential .

• Competing peptide assay: Pre-incubate the antibody with the immunizing peptide before application to demonstrate binding specificity.

• Immunoprecipitation followed by mass spectrometry: This approach confirms that the immunoprecipitated protein is indeed ACO2.

Documented validation results should be maintained according to good laboratory practice guidelines. Remember that antibody performance can vary between lots and may degrade over time, necessitating periodic revalidation .

What are the best practices for using ACO2 antibodies in Western blotting?

Successful Western blotting with ACO2 antibodies requires attention to several methodological details:

• Sample preparation:

  • Use fresh samples and include protease inhibitors in lysis buffers

  • For mitochondrial proteins like ACO2, consider mitochondrial enrichment protocols

  • Load appropriate amounts of protein (typically 20-30 μg of total protein)

• Gel selection:

  • Use 7.5% SDS-PAGE gels for optimal resolution of the 85 kDa ACO2 protein

• Transfer conditions:

  • Optimize transfer time and voltage for efficient transfer of the 85 kDa protein

  • Consider using PVDF membranes for better protein retention

• Blocking and antibody incubation:

  • Test different blocking agents (5% BSA or 5% non-fat milk)

  • Use appropriate antibody dilutions (1:1,000 to 1:10,000 depending on the specific antibody)

  • Include proper washing steps to reduce background

• Controls:

  • Include mitochondrial markers (e.g., VDAC) as loading controls

  • Consider using cytosolic markers (e.g., α-tubulin) to assess fractionation quality

  • Include positive control samples with known ACO2 expression

Following these methodological guidelines will help ensure specific detection of ACO2 and reliable quantification of protein levels in your samples.

How should ACO2 antibodies be stored and handled?

Proper storage and handling of ACO2 antibodies are crucial for maintaining their performance over time:

• Storage conditions:

  • Follow manufacturer's recommendations for storage temperature (typically -20°C for aliquots)

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Store antibody solutions away from direct light

• Working dilutions:

  • Prepare fresh working dilutions for each experiment

  • Dilute in recommended buffers (typically PBS with 0.1% BSA)

  • Consider adding sodium azide (0.02%) to working dilutions if they will be stored

• Quality control:

  • Note the lot number and validate each new lot

  • Track antibody performance over time

  • Re-test antibody activity if stored for extended periods

• Contamination prevention:

  • Use sterile technique when handling antibody solutions

  • Avoid introducing bacteria or other contaminants

By following these practices, researchers can maximize antibody lifespan and ensure consistent experimental results over time.

How can ACO2 antibodies be used to investigate mitochondrial dysfunction in neurodegenerative diseases?

ACO2 mutations have been identified in several neurodegenerative conditions, including infantile cerebellar retinal degeneration (ICRD) and optic neuropathies . ACO2 antibodies serve as valuable tools for investigating these conditions through multi-faceted approaches:

• Protein expression analysis:

  • Compare ACO2 protein levels in patient-derived cells versus controls using Western blotting

  • Correlate expression levels with disease severity and progression

  • Assess the impact of gene variants on protein stability and expression

• Subcellular localization studies:

  • Use immunofluorescence to evaluate ACO2 localization in mitochondria

  • Investigate potential mislocalization in disease states

  • Combine with mitochondrial markers to assess morphological changes

• Integrated functional studies:

  • Complement ACO2 protein detection with enzymatic activity assays

  • Measure mitochondrial aconitase activity in patient-derived cells

  • Correlate protein levels with enzyme activity and mtDNA copy number

• Transcriptomic correlation:

  • Analyze co-expression patterns between ACO2 and other disease-relevant genes

  • Validate protein-level changes of transcriptomically identified candidates like LRP8 and ANK3

  • Develop multiparameter assays to assess pathway dysregulation

Recent research has demonstrated that biallelic ACO2 variants can reduce mitochondrial aconitase activity to approximately 29.3% of wild-type levels, while monoallelic variants result in approximately 69.0% activity . These findings highlight the importance of quantitative protein analysis in understanding genotype-phenotype correlations in ACO2-related disorders.

What protocols are recommended for measuring ACO2 enzyme activity in conjunction with antibody-based detection?

Combining ACO2 antibody detection with enzymatic activity measurement provides a comprehensive assessment of ACO2 function in research and clinical samples:

• Sample preparation for dual analysis:

  • Prepare mitochondrial extracts from peripheral blood leukocytes or cultured cells

  • Divide samples for parallel protein detection and activity assays

  • Maintain consistent sample handling to minimize variability

• Activity assay methodology:

  • Use standardized aconitase activity detection kits

  • Measure spectrophotometrically the conversion of citrate to isocitrate

  • Express activity as a percentage of control samples

• Protein quantification protocol:

  • Perform Western blotting with validated ACO2 antibodies

  • Include appropriate loading controls (e.g., VDAC for mitochondrial content)

  • Use densitometry for quantitative analysis

• Data integration approach:

  • Normalize activity to protein levels to distinguish between expression and functional defects

  • Calculate specific activity (activity per unit of protein)

  • Compare activity-to-expression ratios between experimental groups

Research has shown that in cases with biallelic ACO2 variants, mitochondrial aconitase activity drops to approximately 29.3% of wild-type levels, while the mtDNA copy number decreases to 43.5% . These measurements provide critical insights into the functional consequences of genetic variations and their potential contribution to disease pathogenesis.

How do you troubleshoot non-specific binding when using ACO2 antibodies?

Non-specific binding can complicate interpretation of experimental results when using ACO2 antibodies. Here are methodological approaches to identify and resolve these issues:

• Identifying non-specific binding:

  • Unexpected bands on Western blots (ACO2 should appear at ~85 kDa)

  • Diffuse or unusual staining patterns in immunocytochemistry

  • Positive signal in negative control samples

  • Signal in tissues or cells known not to express ACO2

• Optimization strategies:

  • Blocking optimization: Test different blocking agents (BSA, milk, commercial blockers) and concentrations

  • Antibody dilution: Use higher dilutions of primary antibody (e.g., 1:3,000 instead of 1:1,000)

  • Incubation conditions: Adjust temperature and duration of antibody incubation

  • Washing protocols: Increase wash duration or detergent concentration

• Validation approaches:

  • Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

  • Multiple antibodies: Test different antibody clones targeting distinct epitopes of ACO2

  • Knockdown controls: Use siRNA to reduce ACO2 expression and confirm signal reduction

• Technical considerations:

  • Sample preparation: Ensure complete protein denaturation for Western blotting

  • Fixation methods: Optimize fixation protocols for immunocytochemistry/immunohistochemistry

  • Secondary antibody selection: Choose secondary antibodies with minimal cross-reactivity

By systematically implementing these approaches, researchers can significantly improve specificity and reduce background when using ACO2 antibodies across different experimental applications.

What is the relationship between ACO2 function and iron metabolism, and how can this be studied using antibodies?

ACO2 function is intimately connected to iron metabolism, particularly through its iron-sulfur cluster requirement and relationship with iron regulatory proteins:

• Molecular basis of the ACO2-iron relationship:

  • ACO2 contains an iron-sulfur [4Fe-4S] cluster essential for its enzymatic activity

  • It is functionally related to iron regulatory proteins, particularly IRP1, which can interchangeably bind to iron-responsive elements or possess aconitase activity in its Fe-containing form

  • Molecular docking studies predict that certain ACO2 variants disrupt interactions with the iron-sulfur cluster

• Methodological approaches to study this relationship:

  • Co-immunoprecipitation: Use ACO2 antibodies to pull down protein complexes and analyze iron-related binding partners

  • Double immunolabeling: Combine ACO2 antibodies with antibodies against iron regulatory proteins to assess co-localization

  • Fractionation studies: Compare cytosolic and mitochondrial aconitase activities and protein levels

  • Iron chelation experiments: Assess changes in ACO2 protein levels and activity under iron-depleted conditions

• Combined functional approaches:

  • Measure both ACO2 protein levels and enzyme activity under various iron availability conditions

  • Analyze effects of iron supplementation or chelation on the severity of phenotypes in ACO2-deficient cells

  • Track changes in iron-responsive element-regulated genes alongside ACO2 activity

• Structure-function analysis:

  • Use site-directed mutagenesis to modify iron-binding sites and assess effects on protein stability and function

  • Compare wild-type and variant ACO2 in terms of ligand-binding properties and iron interactions

Understanding this relationship has implications for both basic research and clinical applications, as iron dysregulation and mitochondrial dysfunction are common features in many neurodegenerative diseases associated with ACO2 mutations.

How can transcriptomic and proteomic approaches be integrated with ACO2 antibody-based studies?

Integrating transcriptomic and proteomic approaches with ACO2 antibody-based studies provides a comprehensive understanding of ACO2-related pathways and disease mechanisms:

• Integrated experimental design:

  • Perform parallel transcriptomic and proteomic analyses on the same experimental samples

  • Use ACO2 antibodies for protein validation of transcriptomically identified candidates

  • Apply pathway analysis to identify networks involving ACO2

• Validation of transcriptomic findings:

  • Identify genes whose expression correlates with ACO2 (e.g., LRP8 and ANK3)

  • Validate protein-level changes using specific antibodies against these candidates

  • Assess co-localization with ACO2 using immunofluorescence techniques

• Functional correlation methodologies:

  • Create protein-protein interaction networks based on transcriptomic data

  • Validate key interactions using co-immunoprecipitation with ACO2 antibodies

  • Perform functional assays to confirm the biological relevance of identified interactions

• Disease-specific applications:

  • Compare transcriptomic profiles from patient cells with ACO2 mutations to controls

  • Validate differential expression at the protein level using antibody-based techniques

  • Correlate findings with clinical features and disease severity

Recent research leveraged this integrated approach to identify 80 key candidate genes involved in ACO2-related neuropathy . Among these, LRP8 and ANK3 showed significant positive correlation with ACO2 at the transcriptomic level, which was subsequently validated through protein analysis. This integrated approach revealed that in addition to mitochondrial dysfunction, immune and neurophysiological functions are also involved in ACO2-related pathologies .

How are ACO2 antibodies used in studying genotype-phenotype correlations in ACO2-related disorders?

ACO2 mutations cause a spectrum of neurological disorders with varying severities, making genotype-phenotype correlation studies crucial:

• Patient cohort analysis approaches:

  • Use ACO2 antibodies to assess protein expression in patient-derived cells

  • Compare protein levels between dominant and recessive mutation carriers

  • Correlate expression levels with clinical severity metrics

• Mutation-specific effects assessment:

  • Compare protein levels across different mutation types (missense, truncating, etc.)

  • Assess subcellular localization changes using immunofluorescence

  • Determine if specific domains are differentially affected by various mutations

• Methodological considerations:

  • Standard sample collection and processing protocols to ensure comparability

  • Use of appropriate controls (healthy individuals, carriers, affected individuals)

  • Quantitative image analysis for immunofluorescence studies

Research has revealed important genotype-phenotype correlations in ACO2-related disorders:

• Dominant mutations (found in 50 individuals) typically cause less severe phenotypes than recessive mutations (found in 11 individuals)

• Recessive cases show more severe clinical manifestations, though not necessarily earlier onset

• Extraocular features occur in 27% of recessive cases compared to 11% of dominant cases

These findings highlight how antibody-based protein assessment can contribute to understanding the relationship between genetic variations and clinical presentations.

What protocols are recommended for using ACO2 antibodies in diagnostic applications?

While ACO2 antibodies are primarily research tools, they may have diagnostic value in specialized settings:

• Tissue and sample processing protocols:

  • Standardized fixation methods for tissue samples (10% neutral buffered formalin for 24-48 hours)

  • Consistent antigen retrieval techniques (heat-induced epitope retrieval)

  • Validated dilution ranges for diagnostic applications (typically 1:1,000 for IHC-P)

• Quality control requirements:

  • Inclusion of positive and negative control tissues in each batch

  • Standardized scoring systems for expression levels

  • Regular validation using molecularly confirmed cases

• Complementary testing approaches:

  • Integration with enzymatic activity measurements

  • Correlation with genetic testing results

  • Combined assessment with other mitochondrial markers

• Interpretation guidelines:

  • Differential expression patterns in affected versus unaffected tissues

  • Recognition of subcellular localization changes that may indicate pathology

  • Awareness of potential confounding factors (tissue quality, processing artifacts)

How might ACO2 antibodies contribute to developing therapeutic approaches for ACO2-related disorders?

ACO2 antibodies can facilitate therapeutic development through several research applications:

• Drug screening methodologies:

  • High-throughput screening assays using ACO2 antibodies to detect protein stabilization

  • Assessment of compounds that may enhance residual ACO2 activity

  • Evaluation of therapies targeting downstream pathways identified through ACO2 interactions

• Gene therapy monitoring:

  • Quantification of ACO2 expression following gene therapy interventions

  • Assessment of proper subcellular localization of transgene-expressed protein

  • Long-term monitoring of therapeutic efficacy through protein expression analysis

• Patient stratification approaches:

  • Development of antibody-based assays to identify patients most likely to respond to specific therapies

  • Correlation of baseline protein levels with treatment outcomes

  • Identification of biomarkers for treatment response

• Therapeutic mechanism studies:

  • Investigation of how potential therapeutic compounds affect ACO2 stability and function

  • Assessment of effects on protein-protein interactions and signaling networks

  • Monitoring of changes in mitochondrial morphology and function following treatment

These applications are particularly relevant given recent findings about the molecular mechanisms underlying ACO2-related neuropathies, including alterations in metabolism-related, immune-related, neurophysiological-related, and calcium-related signaling pathways .

What novel applications of ACO2 antibodies are emerging in mitochondrial research?

Emerging applications of ACO2 antibodies in mitochondrial research include:

• Super-resolution microscopy techniques:

  • Nanoscale visualization of ACO2 distribution within mitochondria

  • Assessment of protein clustering and microcompartmentalization

  • Co-localization studies with other Krebs cycle enzymes at unprecedented resolution

• Live-cell imaging applications:

  • Development of cell-permeable ACO2 antibody fragments for live imaging

  • Monitoring of dynamic changes in ACO2 distribution during cellular stress

  • Real-time assessment of mitochondrial responses to metabolic challenges

• Mitochondrial dynamics studies:

  • Investigation of ACO2 distribution during mitochondrial fission and fusion events

  • Analysis of protein redistribution during mitophagy

  • Correlation between ACO2 localization and functional mitochondrial domains

• Multi-omics integration approaches:

  • Combining antibody-based spatial proteomics with metabolomics

  • Correlation of ACO2 distribution with local metabolite concentrations

  • Integration with mitochondrial transcriptomics to map structure-function relationships

These emerging techniques expand the utility of ACO2 antibodies beyond traditional applications, providing new insights into mitochondrial biology and disease mechanisms. Recent research has already demonstrated the value of integrating ACO2 protein studies with transcriptomics and functional assays to identify new pathways involved in mitochondrial diseases .