ACO2 Antibody

What is ACO2 and what is its biological significance?

ACO2, or mitochondrial aconitase, is an 85 kDa protein that plays a key function in cellular energy production through the tricarboxylic acid (TCA) cycle . It catalyzes the stereospecific isomerization of citrate to isocitrate via cis-aconitate in the second step of the TCA cycle. Loss of ACO2 activity has a major impact on cellular and organismal survival, as it is critical for energy production .

Recent studies have shown that ACO2 deficiency increases vulnerability to Parkinson's disease via mitochondrial and autophagic dysfunction . Furthermore, ACO2 has emerged as a potential biomarker for various pathological conditions, including cancer and acute kidney injury (AKI) . The multi-dimensional evidence supports the essential roles of ACO2 in cellular metabolism and disease pathophysiology.

What are the key applications for ACO2 antibodies in research?

ACO2 antibodies are versatile tools with several validated applications:

ACO2 antibodies have been successfully used to detect endogenous protein in various tissues and cell lines, including brain tissue (human, mouse, rat, pig, rabbit), cerebellum tissue, PC-12 cells, HEK-293 cells, and Neuro-2a cells . The wide range of validated applications makes these antibodies valuable for multi-dimensional characterization of ACO2 expression and function.

What species cross-reactivity can be expected from ACO2 antibodies?

Most commercially available ACO2 antibodies demonstrate broad species cross-reactivity. Based on the provided information, ACO2 antibodies typically react with:

• Human

• Mouse

• Rat

• Monkey (Mk)

• Pig

• Rabbit (in brain tissue)

This cross-reactivity is often due to the high conservation of ACO2 protein sequence across mammalian species. For example, the immunogen used for one antibody is "a synthetic peptide corresponding to a sequence at the C-terminus of human Aconitase 2, identical to the related mouse and rat sequences" . This conservation enables comparative studies across different model organisms, facilitating translational research from animal models to human applications.

How should ACO2 antibody specificity be validated for mitochondrial research?

Validating antibody specificity is critical for reliable mitochondrial research involving ACO2. A comprehensive validation approach should include:

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight of 85 kDa in Western blot analysis .

• Knockdown/knockout controls: Perform siRNA-mediated knockdown or CRISPR-Cas9 knockout of ACO2, as demonstrated in research where "ACO2 knockdown or blockade cells showed features of mitochondrial and autophagic dysfunction" . The signal should be significantly reduced or absent in these samples.

• Subcellular fractionation: Since ACO2 is a mitochondrial protein, enrichment in mitochondrial fractions compared to cytosolic fractions confirms specificity.

• Multi-antibody approach: Use at least two different antibodies targeting different epitopes of ACO2 to confirm consistent patterns of expression and localization.

• Species-specific validation: When applying antibodies across species, verify specificity in each species individually, as cross-reactivity may vary despite sequence homology .

• Immunohistochemical pattern analysis: In tissues known to have high mitochondrial content (like brain, heart, liver), ACO2 staining should show the characteristic punctate pattern of mitochondrial proteins with appropriate antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) .

What are the optimal experimental conditions for detecting ACO2 in cancer research?

Cancer research involving ACO2 requires careful optimization of experimental conditions based on recent findings that "ACO2 was highly expressed in most cancers, showing early diagnostic value in six tumor types" .

Western Blotting Optimization:

• Sample preparation: Complete lysis of mitochondria is essential using detergents like RIPA buffer with protease inhibitors

• Loading control selection: Use mitochondrial markers (VDAC, COX IV) alongside traditional housekeeping proteins

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Antibody dilution: Use high dilutions (1:5000-1:50000) for specific ACO2 antibodies to minimize background

Immunohistochemistry for Cancer Tissue:

• Antigen retrieval: Use TE buffer pH 9.0 for optimal ACO2 epitope retrieval

• Signal amplification: Consider tyramide signal amplification for low abundance detection

• Controls: Include both negative controls and tissue with known ACO2 expression patterns

Flow Cytometry for Immune Infiltration Studies:
Given the finding that "ACO2 expression was associated with immune cell infiltration, such as CD8+ T cells and tumor-associated neutrophils, in some cancers" , flow cytometry protocols should:

• Use 0.40 μg of antibody per 10^6 cells for intracellular staining

• Include permeabilization steps optimized for mitochondrial proteins

• Consider co-staining with immune cell markers (CD8, CD4, etc.) to analyze correlations

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

Research has demonstrated that "ACO2 deficiency increases vulnerability to Parkinson's disease via mitochondrial and autophagic dysfunction" , making ACO2 antibodies valuable tools for neurodegenerative disease research.

A comprehensive investigation should include:

• Comparative expression analysis: Use Western blotting to quantify ACO2 protein levels in patient-derived samples versus controls. This approach revealed that "ACO2 activity was significantly decreased in the PBMCs from PD patients" .

• Cellular localization studies: Employ immunofluorescence to examine altered mitochondrial morphology and ACO2 distribution in neuronal models. Changes in mitochondrial network appearance often accompany dysfunction.

• Dual staining protocols: Co-immunostaining of ACO2 with autophagy markers (LC3, p62) to investigate the relationship between mitochondrial dysfunction and autophagic processes. Research shows "the transcription of autophagy-related genes LC3 and Atg5 was significantly downregulated via inhibited histone acetylation at the H3K9 and H4K5 sites" .

• Animal model validation: Assess ACO2 expression in brain regions of animal models (particularly substantia nigra for PD research) using immunohistochemistry with optimized protocols.

• Functional correlation: Combine ACO2 immunostaining with functional mitochondrial assays (membrane potential, ROS production) to correlate protein levels with functional outcomes.

How does ACO2 contribute to cancer progression and immune evasion?

Recent pan-cancer analysis has revealed complex relationships between ACO2, cancer progression, and immune responses :

ACO2 contributes to cancer progression through several mechanisms:

• Metabolic reprogramming: ACO2 alterations affect the TCA cycle, potentially contributing to the Warburg effect, where "cancer cells produce lactic acid from glucose even under non-hypoxic conditions... partly due to the defect in mitochondria TCA cycle" .

• Differential expression patterns: ACO2 is "highly expressed in most cancers, showing early diagnostic value in six tumor types" , suggesting tumor-specific metabolic adaptations.

• Immune microenvironment modulation: ACO2 expression shows significant correlations with tumor-infiltrating immune cells:

  • Positive correlation with B-cell infiltration in breast cancer (BRCA), colorectal adenocarcinoma (COAD), and head and neck squamous cell carcinoma (HNSC)

  • Correlation with CD4+ T cells in HPV-positive HNSC and lung adenocarcinoma (LUAD)

  • Correlation with CD8+ T cells in kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), and LUAD

  • Strong positive correlation with tumor-associated neutrophils (TANs) in several cancers, especially COAD, LIHC, and prostate adenocarcinoma (PRAD)

• Immune checkpoint regulation: "For most types of cancer, there was a significant correlation between ACO2 expression and the levels of immune checkpoint-associated genes" , suggesting ACO2 may influence immune evasion mechanisms.

• Immunotherapy relevance: ACO2 "was positively correlated with multiple immunostimulators in some cancers, such as LIHC, PCPG, and PRAD" , indicating potential as a marker for immunotherapy response prediction.

These findings suggest ACO2 could serve as both a diagnostic biomarker and potential immunotherapy response predictor in specific cancer types.

What is the relationship between ACO2 and acute kidney injury biomarkers?

Research has identified ACO2 as an important biomarker for acute kidney injury (AKI) :

• Gene expression pattern: Through weighted gene co-expression network analysis (WGCNA), ACO2 was identified as one of the top three genes with the most connected nodes in a protein-protein interaction (PPI) network related to AKI, alongside alanine-glyoxylate aminotransferase 2 (AGXT2) and serine hydroxymethyltransferase 1 (SHMT1) .

• Expression changes in AKI: Experimental validation in a rat AKI model confirmed that "the relative mRNA expression and protein levels of AGXT2, SHMT1 and ACO2 showed a decrease in the model group" , supporting their potential as biomarkers.

• Metabolic implications: As a TCA cycle enzyme, reduced ACO2 expression in AKI suggests impaired mitochondrial energy metabolism in kidney tissue during injury.

• Diagnostic application: The study concluded that "there is a close association between AGXT2, SHMT1 and ACO2 genes and the development of AKI, and the down-regulation of their expression may be a potential biomarker for clinical detection of AKI" .

The combination of bioinformatic analysis and experimental validation strengthens the case for ACO2 as part of a biomarker panel for early AKI detection, potentially enabling earlier therapeutic intervention.

What are common technical challenges when using ACO2 antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with ACO2 antibodies:

• Mitochondrial protein extraction issues:

  • Challenge: Incomplete mitochondrial lysis leading to poor ACO2 detection

  • Solution: Use specialized mitochondrial extraction buffers containing digitonin or stronger detergents like CHAPS; sonicate samples briefly to improve extraction

• Non-specific bands in Western blotting:

  • Challenge: Additional bands appearing besides the expected 85 kDa ACO2 band

  • Solution: Optimize antibody dilutions (1:5000-1:50000 is recommended) ; increase washing steps; consider more stringent blocking with 5% BSA instead of milk

• Variable immunohistochemistry results:

  • Challenge: Inconsistent staining patterns in different tissue samples

  • Solution: Standardize antigen retrieval methods (TE buffer pH 9.0 is recommended for ACO2) ; optimize antibody concentration (1:500-1:2000); ensure consistent fixation protocols

• Flow cytometry background issues:

  • Challenge: High background when using ACO2 antibodies for intracellular staining

  • Solution: Use 0.40 μg antibody per 10^6 cells ; optimize permeabilization protocols specifically for mitochondrial proteins; include appropriate isotype controls

• Species cross-reactivity variations:

  • Challenge: Antibody performs differently across species despite sequence homology

  • Solution: Validate each antibody specifically for your species of interest; consider species-specific antibodies when available

• Storage and stability concerns:

  • Challenge: Loss of antibody activity over time

  • Solution: Store antibodies at -20°C in small aliquots to avoid freeze-thaw cycles; for reconstituted antibodies, store at 4°C for up to one month or at -20°C for six months

How should experiments be designed to investigate ACO2's role in Parkinson's disease?

Based on findings that "ACO2 deficiency increases vulnerability to Parkinson's disease via mitochondrial and autophagic dysfunction" , a comprehensive experimental design should include:

• Patient sample analysis:

  • Compare ACO2 protein levels and enzymatic activity in PBMCs from PD patients versus age-matched controls

  • Analyze ACO2 genetic variants, particularly the A252T variant identified in PD patients

• In vitro cellular models:

  • Establish cellular models with ACO2 knockdown or overexpression in neuronal cell lines

  • Create cells expressing specific ACO2 variants (A252T) using CRISPR-Cas9 gene editing

  • Assess:

    • Mitochondrial function (membrane potential, ATP production, ROS levels)

    • Autophagic flux (LC3-II/I ratio, p62 levels)

    • Histone acetylation at H3K9 and H4K5 sites

    • Transcription levels of autophagy-related genes (LC3, Atg5)

• Drosophila models:

  • Generate Drosophila models expressing the A252T variant as described in the literature

  • Evaluate:

    • Motor deficits through climbing assays

    • DA neuron degeneration after toxin exposure (6-OHDA, rotenone)

    • Mitochondrial morphology and function

    • Lifespan and stress resistance

• Therapeutic intervention testing:

  • Test compounds that might "ameliorate energy metabolism by targeting ACO2"

  • Evaluate antioxidants using ACO2 as a biomarker in experimental models

  • Assess whether enhancing mitochondrial function can reverse autophagy defects

• Controls and validation:

  • Include both wild-type controls and disease models targeting other pathways

  • Validate findings across multiple model systems (cells, flies, possibly rodents)

  • Correlate experimental findings with clinical observations

This multi-dimensional approach allows for comprehensive characterization of ACO2's role in PD pathogenesis and potential therapeutic targeting.

What controls are essential when studying ACO2 in cancer immunology research?

Given the finding that "ACO2 expression was associated with immune cell infiltration... in some cancers" , the following controls are essential for cancer immunology research involving ACO2:

• Expression controls:

  • Positive controls: Include tissues/cells known to express high levels of ACO2 (e.g., brain tissue, liver tissue)

  • Negative controls: Use ACO2 knockdown/knockout models or tissues with naturally low ACO2 expression

  • Isotype controls: Include appropriate isotype antibodies to assess non-specific binding

• Cancer type-specific controls:

  • Match tumor and adjacent normal tissue from the same patient

  • Include multiple cancer subtypes when studying pan-cancer effects, as ACO2's relationship with immune infiltration varies by cancer type

• Immune cell infiltration controls:

  • Positive correlation controls: For cancers where ACO2 correlates positively with specific immune cells (e.g., TANs in COAD, LIHC, and PRAD)

  • Negative correlation controls: For cancers where ACO2 shows no correlation with immune infiltration

  • Multi-algorithm validation: Use multiple computational approaches (TIMER, EPIC, CIBERSORT, etc.) as done in the research

• Checkpoint expression controls:

  • Include samples with known high and low expression of immune checkpoint genes

  • Compare correlations across multiple checkpoint molecules, as the research found "significant correlation between ACO2 expression and the levels of immune checkpoint-associated genes"

• Technical controls for co-expression studies:

  • RNA-level controls: Include housekeeping genes for normalization

  • Protein-level controls: Use multiple antibodies targeting different epitopes of ACO2

  • Spatial controls: Assess co-localization versus system-wide expression patterns

• Functional validation controls:

  • Manipulation controls: Compare immune cell behavior in ACO2-high versus ACO2-low conditions

  • Pathway inhibition controls: Use specific inhibitors of pathways identified in KEGG pathway enrichment analysis of ACO2-associated genes

The implementation of these controls will strengthen the validity and reproducibility of findings regarding ACO2's role in cancer immunology.

What emerging applications of ACO2 antibodies should researchers consider exploring?

Based on recent findings, several promising research directions for ACO2 antibodies emerge:

• Cancer immunotherapy biomarker development: Given that ACO2 "could be used as an auxiliary prognostic marker or as a marker for immunotherapy in some tumor types" , researchers should explore its utility in predicting immunotherapy response across different cancer types.

• Neurodegenerative disease early detection: Since "ACO2 activity was significantly decreased in the PBMCs from PD patients" , developing blood-based diagnostic tests using ACO2 antibodies could enable earlier intervention in neurodegenerative diseases.

• Metabolic reprogramming in disease: Investigate how ACO2 alterations affect lipid metabolism, as lipidomics analysis identified "19 significantly altered metabolites, including 17 with reduced levels and 2 with increased levels" in ACO2 knockdown cells.

• Therapeutic monitoring: Explore ACO2 as a biomarker for monitoring the efficacy of "ameliorating energy metabolism... as a potential therapeutic strategy for PD and other neurodegenerative disorders" .

• Multi-omics integration: Combine ACO2 protein analysis with transcriptomics and metabolomics to develop comprehensive disease signatures, particularly for conditions where mitochondrial dysfunction is implicated.