ACO2 Antibody

What are the key specifications of ACO2 antibodies available for research?

ACO2 antibodies are available with various specifications to meet different research needs. Most commonly, these antibodies target different regions of the ACO2 protein and come in different formats based on host and clonality. For example, the ABIN357262 antibody targets the central region of human ACO2, is raised in rabbits as a polyclonal antibody, and demonstrates reactivity with human, rat, and mouse samples . This antibody is suitable for Western Blotting (WB) and Enzyme Immunoassay (EIA) applications.

The table below summarizes the common ACO2 antibody variants available for research:

How should I select an appropriate ACO2 antibody for my specific experimental needs?

When selecting an ACO2 antibody for your research, consider these key factors:

• Experimental application: Different ACO2 antibodies are optimized for specific techniques. For protein detection and quantification, select antibodies validated for Western blotting. For localization studies, choose antibodies validated for immunohistochemistry or immunofluorescence .

• Species compatibility: Ensure the antibody has been validated in your species of interest. The search results indicate that many ACO2 antibodies work with human, rat, and mouse samples, but cross-reactivity varies by antibody .

• Target region specificity: Select antibodies that target relevant regions of the ACO2 protein. For studies involving variants like A252T, ensure your antibody can detect the region containing or affected by this variant .

• Clonality considerations: Polyclonal antibodies offer broader epitope recognition but may have batch-to-batch variability. Monoclonal antibodies provide higher specificity and consistency but may be less sensitive .

• Application-specific validation: Review the validation data provided by manufacturers to ensure the antibody has been thoroughly tested for your specific application.

What are the recommended protocols for using ACO2 antibodies in Western blotting?

For optimal Western blotting results with ACO2 antibodies, follow these methodological guidelines:

• Sample preparation:

  • For whole cell lysates, use RIPA buffer with protease inhibitors

  • For mitochondrial enrichment, consider differential centrifugation protocols

  • Maintain cold conditions throughout to preserve protein integrity

• Electrophoresis parameters:

  • Use 10-12% SDS-PAGE gels appropriate for ACO2's molecular weight (~85 kDa)

  • Load 20-50 μg of total protein per lane

  • Include molecular weight markers spanning 50-100 kDa range

• Transfer conditions:

  • Use PVDF membranes for optimal protein binding

  • Transfer at 100V for 60-90 minutes or 30V overnight at 4°C

  • Verify transfer efficiency with reversible protein staining

• Antibody incubation:

  • Block membranes with 5% non-fat milk or BSA in TBS-T for 1 hour

  • Dilute primary ACO2 antibody according to manufacturer's recommendation (typically 1:500-1:2000)

  • Incubate with primary antibody overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibody (1:2000-1:5000)

• Controls to include:

  • Positive control (tissue with known ACO2 expression, such as liver)

  • Loading control (mitochondrial marker for mitochondrial fractions)

  • ACO2 knockdown samples as negative controls

How can I measure ACO2 activity in patient samples and experimental models?

ACO2 activity measurement is crucial for understanding its functional role in disease states. Research has shown that ACO2 activity is significantly decreased in peripheral blood mononuclear cells (PBMCs) from Parkinson's disease patients and correlates with disease progression . Here are methodological approaches:

• Spectrophotometric assays:

  • Measure the conversion rate of citrate to isocitrate through cis-aconitate

  • Monitor absorbance changes at 240nm

  • Include proper controls and blanks to account for non-enzymatic reactions

• Oxygen consumption measurement:

  • Use a Seahorse XF analyzer to measure oxygen consumption rate (OCR)

  • Compare key parameters: basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity

  • Research shows ACO2 deficiency significantly decreases these parameters in cellular models

• Sample considerations:

  • For patient studies, isolate PBMCs from fresh blood samples

  • Standardize collection and processing to minimize variability

  • For cellular models, use specific ACO2 inhibitors (e.g., trans-aconitate) as controls

• Data interpretation:

  • ACO2 activity positively correlates with age at onset in PD

  • Activity is inversely associated with disease duration

  • Environmental factors like pesticide exposure further reduce activity

What are the known mechanisms linking ACO2 dysfunction to Parkinson's disease?

Research has revealed several crucial mechanisms connecting ACO2 dysfunction to Parkinson's disease pathogenesis:

• Mitochondrial metabolism impairment:

  • ACO2 deficiency reduces OCR and cellular ATP levels

  • This leads to decreased basal respiration, ATP-linked respiration, and maximal respiration

  • Impaired mitochondrial membrane potential, especially under oxidative stress conditions

• Autophagy dysregulation:

  • ACO2 deficiency inhibits autophagy flux

  • Decreased LC3-II expression and increased p62 levels

  • Reduced transcription of autophagy-related genes (LC3, Atg5)

  • This occurs via inhibited histone acetylation at H3K9 and H4K5 sites

• α-synuclein interaction:

  • α-synuclein directly binds to ACO2

  • This interaction promotes progressive mitochondrial dysfunction

  • Establishes a direct link between α-synuclein pathology and TCA cycle impairment

• Genetic susceptibility:

  • Rare coding variants (e.g., A252T) lead to decreased aconitase activity

  • In mouse and Drosophila models, these variants cause:

    • Aggravated motor deficits

    • Increased DA neuron degeneration when exposed to neurotoxins (6-OHDA, rotenone)

These mechanisms collectively contribute to increased oxidative stress, energy deficiency, and neuronal vulnerability characteristic of Parkinson's disease.

How does the ACO2 A252T variant affect cellular and molecular functions?

The ACO2 A252T variant, identified in Parkinson's disease patients, demonstrates several functional alterations at cellular and molecular levels:

• Enzymatic activity:

  • Decreased aconitase activity

  • Reduced intracellular ATP production

  • Impaired TCA cycle function

• Mitochondrial respiration:

  • Significantly decreased oxygen consumption rate (OCR)

  • Reduced basal respiration, ATP-linked respiration, and maximal respiration

  • Lower spare respiratory capacity

• Mitochondrial membrane potential:

  • Increased vulnerability to depolarization

  • Enhanced sensitivity to oxidative stressors like rotenone

• Autophagy function:

  • Inhibited autophagic flux

  • Decreased LC3-II and increased p62 levels

  • Reduced expression of autophagy-related genes

• Epigenetic regulation:

  • Inhibited histone acetylation at H3K9 and H4K5 sites

  • This affects transcription of key autophagy genes

• Disease model effects:

  • In knock-in mouse and Drosophila models, the A252T variant led to:

    • Motor deficits and behavioral impairments

    • Enhanced vulnerability to neurotoxins

    • Increased dopaminergic neuron degeneration

These findings establish ACO2 A252T as a risk factor that increases vulnerability to Parkinson's disease, particularly when combined with environmental stressors.

What experimental approaches can demonstrate the interaction between α-synuclein and ACO2?

To investigate the critical interaction between α-synuclein and ACO2, researchers can employ several experimental strategies:

• In vivo models:

  • AAV9-mediated expression of ACO2-EGFP, ACO2-A252T-EGFP, or control vectors in mouse striatum

  • This approach allows for visualization and functional assessment of ACO2 variants in the context of α-synuclein pathology

  • Behavioral assessments can correlate molecular findings with functional outcomes

• Co-localization studies:

  • Immunofluorescence microscopy using antibodies against ACO2 and α-synuclein

  • Super-resolution microscopy for detailed interaction analysis

  • Proximity ligation assays to confirm direct interaction in tissue samples

• Protein interaction analysis:

  • Co-immunoprecipitation with ACO2 antibodies to pull down α-synuclein or vice versa

  • FRET (Förster Resonance Energy Transfer) to assess direct protein interactions in living cells

  • Surface plasmon resonance to measure binding kinetics between purified proteins

• Functional consequence assessment:

  • Measure ACO2 activity in the presence of recombinant α-synuclein

  • Assess mitochondrial function parameters with combined ACO2/α-synuclein manipulation

  • Metabolite profiling to identify TCA cycle alterations resulting from the interaction

Research has demonstrated that targeting the α-synuclein-ACO2 interaction may represent a promising therapeutic strategy for improving mitochondrial function in Parkinson's disease .

How can ACO2 antibodies be used to investigate mitochondrial dysfunction in disease models?

ACO2 antibodies serve as powerful tools for investigating mitochondrial dysfunction across multiple experimental platforms:

• Protein expression profiling:

  • Western blotting to quantify ACO2 levels in different tissues, disease states, or treatment conditions

  • Comparison of ACO2 expression between wild-type and variant forms (e.g., A252T)

  • Assessment of post-translational modifications affecting ACO2 function

• Tissue and cellular localization:

  • Immunohistochemistry to map ACO2 distribution in brain regions affected by neurodegeneration

  • Immunofluorescence co-localization with mitochondrial markers to assess mitochondrial integrity

  • Subcellular fractionation followed by immunoblotting to track ACO2 distribution

• Complex experimental paradigms:

  • ACO2 detection in models exposed to environmental toxins (rotenone, 6-OHDA)

  • Time-course studies to track ACO2 changes during disease progression

  • Evaluation of therapeutic interventions targeting mitochondrial function

• Correlation studies:

  • Pairing ACO2 antibody detection with functional assays:

    • Oxygen consumption rate measurements

    • ATP production assays

    • Mitochondrial membrane potential assessment

  • These correlations help establish causality between ACO2 alterations and functional consequences

• Therapeutic evaluation:

  • Monitoring ACO2 expression and localization following experimental therapies

  • Assessing restoration of normal ACO2 function after genetic interventions (e.g., AAV9-ACO2 delivery)

What are the emerging therapeutic approaches targeting ACO2 in neurodegenerative diseases?

Recent research has identified several promising therapeutic strategies targeting ACO2 for neurodegenerative disorders:

• Gene therapy approaches:

  • AAV9-mediated delivery of wild-type ACO2-EGFP to the striatum has been tested in mouse models

  • This approach can potentially restore normal ACO2 function in affected brain regions

  • Comparative studies between wild-type ACO2 and variant forms (ACO2-A252T) help determine therapeutic efficacy

• Mitochondrial function enhancement:

  • Targeting the interaction between α-synuclein and ACO2 to prevent mitochondrial dysfunction

  • Development of small molecules that can stabilize ACO2 structure and function

  • Antioxidant strategies to protect ACO2's iron-sulfur clusters from oxidative damage

• Autophagy modulation:

  • Since ACO2 deficiency impairs autophagy via histone acetylation mechanisms, targeting these epigenetic pathways represents a potential approach

  • Histone deacetylase inhibitors might counteract the reduced H3K9 and H4K5 acetylation caused by ACO2 dysfunction

  • Autophagy activators could potentially bypass the ACO2-related defects

• Biomarker applications:

  • ACO2 activity in PBMCs shows potential as a biomarker for:

    • Disease progression monitoring

    • Therapeutic response assessment

    • Identification of at-risk individuals

  • This could enable earlier intervention and personalized treatment approaches

These emerging strategies highlight ACO2 as a promising therapeutic target for improving mitochondrial function in Parkinson's disease and potentially other neurodegenerative disorders.

How should researchers interpret discrepancies between ACO2 protein levels and enzyme activity?

Understanding the relationship between ACO2 protein expression and enzymatic activity is crucial for accurate data interpretation:

• Mechanistic explanations for discrepancies:

  • ACO2 contains iron-sulfur clusters that are highly sensitive to oxidative stress

  • Protein may remain intact while the enzyme becomes catalytically inactive

  • Post-translational modifications can affect activity without changing expression levels

• Interpretation framework:

  • Decreased activity with normal expression: Suggests functional inactivation, possibly due to oxidative damage or α-synuclein binding

  • Decreased expression and activity: Indicates transcriptional/translational downregulation

  • Increased expression with normal/low activity: Suggests compensatory upregulation in response to functional deficits

• Methodological considerations:

  • Always measure both protein levels (via immunoblotting) and enzymatic activity

  • Include positive controls with known ACO2 activity

  • Consider cellular fractionation to ensure accurate assessment of mitochondrial ACO2

• Disease-specific patterns:

  • In Parkinson's disease, research shows decreased ACO2 activity in PBMCs correlates with disease parameters

  • Activity changes may precede detectable changes in protein levels

  • Environmental factors known to increase PD risk further reduce ACO2 activity

• Research implications:

  • Therapeutic approaches should address both expression and activity

  • Activity measurements may be more sensitive biomarkers than protein levels

  • Combined analysis provides more comprehensive understanding of ACO2's role in disease pathogenesis

What controls and normalization strategies are essential when using ACO2 antibodies in complex disease models?

Proper controls and normalization strategies are critical for generating reliable and reproducible results with ACO2 antibodies:

• Essential experimental controls:

  • Positive tissue controls: Liver, heart, or brain tissues with known ACO2 expression

  • Negative controls: ACO2 knockdown or knockout samples where available

  • Antibody controls: Secondary antibody-only controls to detect non-specific binding

  • Loading controls: Mitochondrial markers (e.g., VDAC, COX IV) for mitochondrial preparations

• Disease model-specific controls:

  • Age-matched controls to account for age-related changes in mitochondrial function

  • Genetic background controls in transgenic models

  • Vehicle controls in toxin-induced models (e.g., 6-OHDA, rotenone)

• Normalization strategies:

  • For Western blotting: Normalize to mitochondrial housekeeping proteins rather than total cellular proteins

  • For enzymatic activity: Express as activity per unit of mitochondrial protein

  • For immunohistochemistry: Use consistent anatomical regions for quantification

• Validation approaches:

  • Use multiple antibodies targeting different epitopes of ACO2

  • Confirm key findings with complementary techniques (e.g., mass spectrometry)

  • Include genetic rescue experiments to verify specificity of observed phenotypes

• Statistical considerations:

  • Account for biological variability in ACO2 expression and activity

  • Use appropriate statistical tests based on data distribution

  • Report both statistical significance and effect sizes

These strategies help ensure that changes observed in ACO2 expression or function are genuine and disease-relevant rather than artifacts of experimental variation.