ACO3 Antibody

What is ACO3 and what functions does it serve in biological systems?

ACO3 refers to two distinct proteins depending on the biological context. In humans, ACO3 is another term for iron responsive element binding protein 2 (IREB2), an RNA-binding protein that interacts with iron-responsive elements (IRES) . These IRES are stem-loop structures found in the 5'-UTR of ferritin and delta aminolevulinic acid synthase mRNAs, as well as in the 3'-UTR of transferrin receptor mRNA . The human ACO3/IREB2 protein consists of 963 amino acid residues with a molecular mass of approximately 105.1 kilodaltons and is primarily localized in the cytoplasm .

In plants, particularly Arabidopsis, ACO3 (ACONITASE 3) serves as a key component in stress regulation pathways . Plant ACO3 is induced by mitochondrial dysfunction and contributes significantly to stress tolerance mechanisms . The protein undergoes phosphorylation at Ser91, which appears to be critical for its role in stress signaling and response mechanisms .

What are the primary research applications for ACO3 antibodies?

ACO3 antibodies have several important research applications across different experimental techniques. Western Blot analysis represents the most common application for ACO3 antibodies, allowing researchers to detect and quantify ACO3 protein expression in various tissue and cell extracts . ELISA (Enzyme-Linked Immunosorbent Assay) provides quantitative measurement of ACO3 protein levels in solution, offering high sensitivity for protein detection . Immunohistochemistry enables visualization of ACO3 distribution within tissue sections, providing valuable data on protein localization within biological contexts . Immunofluorescence techniques allow researchers to examine subcellular localization of ACO3 and potential co-localization with other proteins of interest.

How do ACO3 antibodies differ between human and plant research applications?

Antibodies targeting human ACO3 (IREB2) are designed to recognize epitopes specific to the 105.1 kDa protein that functions in iron homeostasis regulation . These antibodies are typically validated for applications with human, mouse, and rat samples, as indicated by commercial antibody reactivity information .

In contrast, antibodies for plant ACO3 are specifically developed to target the Arabidopsis ACONITASE 3 protein involved in stress response pathways . These antibodies enable researchers to study phosphorylation status at key residues like Ser91, which plays a crucial role in the protein's function during stress conditions . Plant-specific ACO3 antibodies are essential for investigating how this protein contributes to tolerance against stressors such as ultraviolet B radiation or antimycin A-induced mitochondrial dysfunction .

How can ACO3 antibodies be utilized to study post-translational modifications?

ACO3 antibodies designed to detect specific post-translational modifications (PTMs) provide crucial insights into regulatory mechanisms. In plant systems, phosphorylation of ACO3 at Ser91 is particularly significant, as this modification appears to regulate the protein's function during stress responses . Research indicates that phosphorylation at this site increases under stress conditions and requires signaling through the ANAC017 transcription factor pathway .

For studying phosphorylation states, researchers should employ phospho-specific antibodies that selectively recognize ACO3 only when modified at Ser91. These specialized antibodies enable tracking of phosphorylation dynamics in response to various stressors. Complementary techniques such as phosphatase treatments followed by Western blotting with total ACO3 antibodies can confirm phosphorylation status. Mass spectrometry-based proteomics represents another powerful approach, as demonstrated in research that revealed increased abundance and phosphorylation of ACO3 under stress conditions in plant systems .

What role does ACO3 play in mitochondrial dysfunction signaling pathways?

Research using ACO3 antibodies has revealed that ACO3 functions as both a target and mediator of mitochondrial dysfunction signaling in Arabidopsis . Studies demonstrate that ACO3 abundance and phosphorylation increase under stress conditions, with this response requiring signaling through the ANAC017 transcription factor pathway .

Phosphomimetic mutation experiments at ACO3-Ser91 have shown that the accumulation of phosphorylated ACO3 promotes expression of genes related to mitochondrial dysfunction . Additionally, functional studies reveal that ACO3 contributes significantly to plant tolerance against stressors that induce mitochondrial dysfunction, including ultraviolet B radiation and antimycin A treatment . These findings collectively position ACO3 as a critical component in stress tolerance mechanisms. Researchers investigating mitochondrial signaling pathways should consider using ACO3 antibodies to monitor both total protein levels and phosphorylation status as indicators of stress response activation.

How do cell-based assays compare with traditional methods for detecting specific antibodies?

While not directly related to ACO3 antibodies, research on nicotinic acetylcholine receptor antibodies provides valuable comparative methodology insights. A recent study demonstrated that cell-based assays (CBAs) offer superior specificity compared to traditional radioimmunoprecipitation assays (RIPA) for detecting certain antibodies in autoimmune conditions .

The study showed that CBAs effectively identified clinically relevant antibodies while eliminating false positives that were detected at low levels by RIPA . This methodology distinction is important for researchers developing detection systems for ACO3 antibodies, particularly when studying autoimmune responses or when antibody specificity is critical to experimental outcomes. When designing experiments to detect ACO3 autoantibodies in clinical samples, researchers should consider implementing cell-based assays with cells expressing ACO3 on their surface to enhance specificity and reduce false-positive results.

What optimization strategies improve Western blot results with ACO3 antibodies?

Successful Western blotting with ACO3 antibodies requires careful optimization of several parameters. Sample preparation should include appropriate protease inhibitors to prevent degradation of the 105.1 kDa ACO3 protein . For optimal separation of this relatively large protein, researchers should use lower percentage (7-8%) polyacrylamide gels and extend transfer times to ensure complete protein transfer.

Blocking conditions significantly impact antibody performance; researchers should test different blocking agents (BSA vs. non-fat milk) as some antibodies perform better with specific blockers. Antibody concentration requires systematic titration; starting with manufacturer-recommended dilutions (typically 1:1000) and adjusting based on signal-to-noise ratio. Extended primary antibody incubation (overnight at 4°C) often improves detection of lower abundance targets. For visualization, enhanced chemiluminescence systems offer good sensitivity, but fluorescent secondary antibodies provide better quantitative analysis capabilities and wider linear range.

What controls are essential when working with ACO3 antibodies?

Implementing proper controls is crucial for generating reliable data with ACO3 antibodies. Positive controls should include samples known to express ACO3 protein, such as specific cell lines or tissues with verified expression . Negative controls might include ACO3/IREB2 knockout or knockdown samples, or tissues known not to express the protein.

To verify antibody specificity, pre-adsorption controls using the immunizing peptide can confirm that binding is epitope-specific. Secondary antibody-only controls help identify potential non-specific binding of the secondary antibody. Loading controls (housekeeping proteins like β-actin, GAPDH, or tubulin) are essential for normalizing ACO3 signal between samples, particularly in comparative studies. For phosphorylation studies, phosphatase-treated samples serve as important controls to confirm phospho-antibody specificity.

How can researchers validate the specificity of commercial ACO3 antibodies?

Validating antibody specificity is critical for generating reliable research data. Western blot validation should demonstrate a single band at the expected molecular weight (approximately 105.1 kDa for human ACO3/IREB2) . Multiple bands may indicate non-specific binding or protein degradation. Researchers should compare results across multiple antibodies targeting different epitopes of the same protein to build confidence in specificity.

Genetic validation using siRNA knockdown, CRISPR knockout, or overexpression systems provides compelling evidence of antibody specificity. Correlation between protein levels detected by the antibody and mRNA expression data can provide additional validation. Immunoprecipitation followed by mass spectrometry analysis offers definitive confirmation that the antibody is capturing the intended target. Cross-reactivity testing across species or closely related proteins should be performed if the antibody will be used in comparative studies.

How are ACO3 antibodies employed in studying iron metabolism pathways?

ACO3 antibodies provide valuable tools for investigating iron metabolism, as the human ACO3/IREB2 protein binds to iron-responsive elements in mRNAs encoding proteins involved in iron homeostasis . These antibodies enable researchers to track changes in IREB2 expression and localization in response to altered iron levels or oxidative stress.

Immunoprecipitation with ACO3 antibodies followed by RNA immunoprecipitation (RIP) allows identification of mRNA targets bound by the protein under various conditions. Co-immunoprecipitation experiments help reveal interaction partners that may regulate IREB2 function or be regulated by it in iron metabolism pathways. Western blotting with ACO3 antibodies enables quantitative assessment of protein expression changes in response to iron chelation, supplementation, or in disease models with disrupted iron homeostasis. Immunofluorescence applications provide insights into potential translocation of IREB2 between subcellular compartments under different iron conditions.

What role do multiplexed autoantibody profiling systems play in current research?

While not specifically focused on ACO3 antibodies, recent advances in multiplexed autoantibody profiling provide important methodological context for researchers. A study on urothelial carcinoma patients employed a SeroTag immuno-oncology discovery array to quantify autoantibody reactivity toward 1132 antigens simultaneously . This approach required minimal serum volume while generating comprehensive autoantibody profiles.

The study revealed distinct autoantibody signatures associated with cancer versus healthy controls, treatment response patterns, and development of immune-related adverse events . Similar multiplexed approaches could be applied to study ACO3 autoantibodies in various disease contexts, particularly in conditions involving disrupted iron metabolism or mitochondrial dysfunction. Researchers could employ such systems to evaluate ACO3 autoantibodies alongside other potential biomarkers, creating comprehensive profiles that might reveal unexpected associations or patterns.

What strategies address common challenges when using ACO3 antibodies in immunohistochemistry?

Successful immunohistochemistry (IHC) with ACO3 antibodies requires careful optimization of several critical parameters. Fixation methods significantly impact epitope accessibility; while formalin fixation is standard, researchers should consider testing alternative fixatives if signal is weak. Antigen retrieval is often critical; heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be systematically tested.

Background reduction requires optimization of blocking solutions (e.g., 5-10% normal serum from the species of secondary antibody origin). Antibody dilution should be determined empirically, starting with manufacturer recommendations and adjusting based on signal-to-noise ratio. For visualization, amplification systems such as polymer-based detection can enhance sensitivity for low-abundance targets. Positive and negative tissue controls must be included in every experiment to validate staining patterns and reagent performance.

How can researchers quantitatively assess ACO3 expression levels using antibody-based methods?

Quantitative assessment of ACO3 expression requires carefully optimized protocols and appropriate controls. For Western blot quantification, researchers should ensure samples are within the linear range of detection and use fluorescent secondary antibodies rather than chemiluminescence for more accurate quantification . Normalization to housekeeping proteins is essential, but choice of reference protein should be validated for stability across experimental conditions.

ELISA provides direct quantification if standard curves are generated using purified recombinant ACO3 protein. For immunohistochemistry quantification, digital image analysis using appropriate software can measure staining intensity across tissue sections, though standardization between batches remains challenging. Flow cytometry offers single-cell resolution for quantifying ACO3 in cell populations, particularly useful when studying heterogeneous samples. Mass cytometry or imaging mass cytometry can provide highly multiplexed quantification of ACO3 alongside dozens of other proteins at single-cell resolution.

How might emerging antibody technologies enhance ACO3 research?

Emerging technologies promise to expand the capabilities of ACO3 antibody applications. Single-domain antibodies (nanobodies) derived from camelid antibodies offer smaller size for accessing restricted epitopes and superior penetration in tissue samples. These could provide new insights into ACO3 localization and interaction partners. Recombinant antibody fragments with site-specific conjugation capabilities enable precise control over labeling ratio and position, enhancing imaging and quantification applications.

Bispecific antibodies targeting ACO3 and an interaction partner simultaneously could facilitate studies of protein-protein interactions in situ. Antibody-DNA conjugates compatible with spatial transcriptomics would allow correlation between ACO3 protein localization and local transcriptional profiles. Advances in cryo-electron microscopy compatible immunolabeling might reveal structural details of ACO3 in its native cellular environment or in complex with binding partners.

What research gaps remain in understanding ACO3 function across different biological systems?

Despite progress in ACO3 research, significant knowledge gaps remain. The relationship between plant ACO3 and human ACO3/IREB2 requires further comparative studies to determine functional conservation and divergence . The role of ACO3 phosphorylation in plants has been partially characterized, but the equivalent post-translational modifications in human ACO3/IREB2 and their functional significance remain understudied .

Potential roles of ACO3 in disease pathogenesis, particularly conditions involving mitochondrial dysfunction or iron dysregulation, represent important areas for investigation. ACO3 interaction networks in different cellular contexts and how these interactions change during stress responses or disease states need comprehensive mapping. The mechanisms by which ACO3 contributes to stress tolerance in plants could inform therapeutic approaches targeting related pathways in human diseases . Development of more specific tools, including monoclonal antibodies targeting distinct epitopes or post-translational modifications, would accelerate progress in addressing these research gaps.