ALDOA Antibody
Description
Introduction to ALDOA Antibody
ALDOA antibody is an immunoglobulin specifically designed to recognize and bind to Aldolase A (fructose-bisphosphate aldolase A), a key enzyme in the glycolytic pathway. Aldolase A catalyzes the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, playing a crucial role in cellular energy metabolism . While primarily known for its enzymatic function, recent research has uncovered non-glycolytic roles of ALDOA, including interactions with cytoskeletal components that influence cell mobility and metastatic potential in cancer .
ALDOA is predominantly found in developing embryos and is produced in even greater amounts in adult muscle. Its expression is repressed in adult liver, kidney, and intestine, while its levels in brain and other nervous tissues are comparable to those of aldolase C . ALDOA belongs to the class I fructose-bisphosphate aldolase family and is encoded by the ALDOA gene, which produces a homotetrameric protein structure .
Types and Sources
ALDOA antibodies are available in both polyclonal and monoclonal formats, each with distinct characteristics:
| Antibody Type | Host Species | Clonality | Advantages |
|---|---|---|---|
| Polyclonal | Rabbit, Goat | Polyclonal | Recognizes multiple epitopes, stronger signal |
| Monoclonal | Mouse | Monoclonal | High specificity, consistent performance |
Polyclonal antibodies are typically generated by immunizing animals (commonly rabbits or goats) with recombinant ALDOA protein or synthetic peptides corresponding to specific regions of the ALDOA sequence . For instance, some commercially available antibodies target the N-terminal region (amino acids 66-95) of human ALDOA .
Monoclonal antibodies offer greater specificity and are produced using hybridoma technology to ensure consistent performance across different batches .
Reactivity and Specificity
Commercial ALDOA antibodies vary in their species reactivity profiles:
The observed molecular weight of ALDOA in western blotting typically ranges from 35-40 kDa, although the calculated molecular weight based on amino acid sequence is approximately 39.4 kDa . Some antibodies detect a higher molecular weight band around 111 kDa, which may represent tetrameric forms or post-translationally modified variants of the protein .
Applications of ALDOA Antibody
ALDOA antibodies are utilized in a wide range of experimental techniques, with specific recommended dilutions for each application:
Western Blotting (WB)
Western blotting remains one of the most common applications for ALDOA antibodies, with recommended dilutions ranging from 1:500 to 1:50,000 depending on the specific antibody and experimental conditions . The technique allows for detection of ALDOA protein in cell lysates and tissue extracts, with reported positive detection in various cell lines including HeLa, HEK-293, HepG2, Jurkat, and tissue samples from multiple species .
Immunohistochemistry (IHC)
ALDOA antibodies are frequently used for immunohistochemical staining of tissue sections, with typical dilutions ranging from 1:50 to 1:2,000 . Many antibodies have been validated on human lung cancer tissue and normal tissues, making them valuable tools for studying ALDOA expression patterns in different physiological and pathological contexts .
Immunofluorescence (IF) and Immunocytochemistry (ICC)
For cellular localization studies, ALDOA antibodies can be used in IF and ICC applications with recommended dilutions typically ranging from 1:10 to 1:800 . These techniques enable visualization of ALDOA distribution within cells and tissues, providing insights into its subcellular localization and potential interactions with other cellular components.
Other Applications
Additional applications for ALDOA antibodies include:
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Immunoprecipitation (IP): Used to isolate ALDOA and its interacting partners
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Co-immunoprecipitation (CoIP): Employed to study protein-protein interactions involving ALDOA
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RNA immunoprecipitation (RIP): Used to investigate RNA-protein interactions
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ELISA: Applied for quantitative detection of ALDOA in biological samples
ALDOA and Cancer Research
ALDOA antibodies have played a crucial role in elucidating the involvement of ALDOA in cancer progression, particularly in lung cancer. Research has demonstrated that ALDOA promotes lung cancer metastasis through a non-glycolytic mechanism involving its interaction with γ-actin .
A study published in Cancer Research utilized ALDOA antibodies to reveal that ALDOA forms a cancer-associated protein-protein interaction with γ-actin, which is independent of its glycolytic function. This interaction promotes actin polymerization and enhances the metastatic potential of lung cancer cells. Importantly, this interaction was specific to cancer cells and not observed in normal cells, making it a potential therapeutic target .
Therapeutic Targeting of ALDOA Interactions
Researchers have identified that disrupting the ALDOA-γ-actin interaction can inhibit cancer metastasis. A designed peptide targeting amino acids 286-302 of ALDOA structure effectively blocked this interaction and reduced the metastatic ability of lung cancer cells without affecting ALDOA's enzymatic activity or causing cytotoxicity .
Furthermore, through virtual screening of approved drugs, raltegravir (RAL) was identified as a small-molecule inhibitor that can disrupt the ALDOA-γ-actin interaction. This finding presents a potential repurposing opportunity for this HIV treatment drug in cancer therapy .
Anti-ALDOA Antibodies as Biomarkers
Recent studies have identified autoantibodies against ALDOA (ALDOA-Abs) in patient serum as potential biomarkers for various medical conditions :
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Myasthenia Gravis: A novel autoantibody targeting ALDOA was identified in patients with myasthenia gravis, particularly in seronegative cases. The autoantibody was detected using tissue-based assays, and ALDOA was confirmed as the target antigen through immunoprecipitation, mass spectrometry, and Western blot techniques .
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Cerebrovascular Disorders: Anti-ALDOA antibodies have been associated with transient ischemic attack (TIA) and acute cerebral infarction (aCI). A study found that patients with TIA or aCI had significantly higher ALDOA-Ab levels compared to healthy donors, with odds ratios of 2.46 (p = 0.0050) for TIA and 2.50 (p < 0.01) for aCI risk .
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Cardiovascular Risk Factors: ALDOA-Abs show strong associations with hypertension, coronary heart disease, and habitual smoking. These antibody levels also correlate well with maximum intima-media thickness, which reflects atherosclerotic stenosis .
The presence of these autoantibodies was confirmed through Western blotting, where GST-ALDOA proteins reacted with serum antibodies from patients, but not with the GST domain alone .
Diagnostic Potential
The multivariate logistic regression analyses from clinical studies demonstrate that ALDOA-Abs can serve as independent predictors of certain medical conditions . This suggests potential applications in developing diagnostic assays for:
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Identifying patients at risk for cerebrovascular events
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Diagnosing seronegative myasthenia gravis
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Risk stratification in patients with cardiovascular risk factors
Future Perspectives in ALDOA Antibody Research
The expanding understanding of ALDOA's multifaceted roles beyond glycolysis opens new avenues for ALDOA antibody applications in both research and clinical settings. Several promising directions include:
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Development of therapeutic antibodies: Based on the understanding of ALDOA's role in cancer progression, therapeutic antibodies specifically targeting the ALDOA-γ-actin interaction domain could potentially inhibit metastasis without affecting normal glycolytic functions .
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Improved diagnostic tools: Standardized assays measuring anti-ALDOA autoantibodies in patient serum could enhance early detection of cerebrovascular disorders and autoimmune conditions like myasthenia gravis .
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Combination therapies: Research into combining ALDOA-targeted approaches with existing treatments may lead to more effective therapeutic strategies, particularly in cancer .
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Structure-function studies: More detailed investigations into the structural basis of ALDOA's non-glycolytic functions could reveal additional therapeutic targets and applications .
Product Specs
Q&A
What is ALDOA and what cellular functions does it perform?
ALDOA (aldolase A, fructose-bisphosphate) is a 39 kDa glycolytic enzyme that functions in the reversible cleavage of fructose-bisphosphate (FBP) to glyceraldehyde-3-phosphate (G-3-P) and dihydroxyacetone phosphate (DHAP) . It is predominantly expressed in skeletal muscle and red blood cells, though it is also found in various other cell types. ALDOA plays a critical role in glucose metabolism through the glycolytic pathway, and recent research has implicated it in cancer progression, particularly in colorectal tumorigenesis and metastasis . Beyond its metabolic functions, ALDOA has been shown to interact with signaling pathways including the AMPK pathway, which affects YAP activity and subsequently influences cell proliferation and migration .
What types of ALDOA antibodies are currently available for research applications?
Researchers have access to both polyclonal and monoclonal ALDOA antibodies, each with distinct advantages depending on the experimental application:
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Polyclonal antibodies: Available from vendors such as Proteintech (catalog #11217-1-AP), these are purified through antigen affinity methods and demonstrate reactivity with human, mouse, and rat samples .
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Monoclonal antibodies: Available from vendors such as Boster Bio (Picoband® monoclonal, clone 6H8, catalog #M05022-2), these offer high specificity for human ALDOA and are recommended for applications requiring minimal background signal .
Both antibody types have been validated across multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunoprecipitation (IP), and Flow Cytometry .
What are the recommended dilutions for different ALDOA antibody applications?
Optimal dilution ratios vary significantly depending on the specific application and the antibody source. Based on validated protocols, the following dilutions are recommended for polyclonal antibodies:
| Application | Recommended Dilution |
|---|---|
| Western Blot (WB) | 1:5000-1:50000 |
| Immunoprecipitation (IP) | 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate |
| Immunohistochemistry (IHC) | 1:100-1:400 |
| Immunofluorescence (IF)/ICC | 1:50-1:500 |
These dilutions may need adjustment based on sample type, detection method, and specific research requirements . Additionally, it is advisable to perform dilution optimization experiments when working with new antibody lots or sample types.
How should I optimize antigen retrieval for IHC with ALDOA antibodies?
Effective antigen retrieval is critical for successful ALDOA detection in tissue sections. The following methodological approach is recommended:
For paraffin-embedded sections, heat-mediated antigen retrieval has shown optimal results using either:
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TE buffer (pH 9.0) as the primary recommendation for polyclonal antibodies like 11217-1-AP .
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EDTA buffer (pH 8.0) for monoclonal antibodies, as demonstrated in validated protocols with the M05022-2 antibody .
For fixed cell preparations, enzyme antigen retrieval using IHC enzyme antigen retrieval reagent (e.g., AR0022) for 15 minutes has produced reliable results in immunocytochemical applications .
When using citrate buffer (pH 6.0) as an alternative, extend the incubation time to ensure adequate epitope exposure. Following antigen retrieval, always block with appropriate serum (typically 10% goat serum) to minimize non-specific binding .
What controls should I include when working with ALDOA antibodies?
Implementing appropriate controls is essential for result validation and troubleshooting:
Positive controls:
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For WB: Use lysates from HEK-293, HeLa, A549, Jurkat, HepG2, MCF-7, or NIH/3T3 cells, which have been validated to express detectable ALDOA levels .
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For IHC: Human skeletal muscle tissue, liver cancer tissue, breast cancer tissue, or rectal cancer tissue sections have demonstrated reliable ALDOA expression .
Negative controls:
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Primary antibody omission: Process samples with all reagents except the primary ALDOA antibody.
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Isotype controls: For monoclonal antibodies, particularly in flow cytometry applications, include an appropriate isotype-matched control antibody (e.g., mouse IgG at 1μg/1×10^6 cells) to identify non-specific binding .
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ALDOA-knockdown cells: When available, cells with ALDOA expression silenced via shRNA provide an excellent specificity control .
How can I validate ALDOA knockdown or overexpression in experimental models?
When establishing ALDOA-manipulated cell lines for functional studies, validation at both transcript and protein levels is essential:
For ALDOA knockdown validation:
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Transcript level: Perform qRT-PCR to quantify ALDOA mRNA reduction compared to control-shRNA transfected cells.
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Protein level: Conduct Western blot analysis using validated ALDOA antibodies (1:5000-1:50000 dilution) to confirm reduced protein expression .
For ALDOA overexpression validation:
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Transcript level: Use qRT-PCR to confirm increased ALDOA mRNA in Lenti-ALDOA transfected cells versus empty vector controls.
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Protein level: Western blot analysis should show significantly increased ALDOA protein in overexpression models .
As demonstrated in colorectal cancer research, successful ALDOA genetic manipulation produces significant and measurable differences in both transcript and protein levels compared to control groups .
How can ALDOA antibodies be used to investigate subcellular localization and pathway interactions?
ALDOA antibodies are powerful tools for investigating protein-protein interactions and subcellular distribution:
For subcellular localization studies:
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Immunofluorescence approach: Use ALDOA antibodies at 1:50-1:500 dilution followed by fluorophore-conjugated secondary antibodies (e.g., DyLight®488 Conjugated anti-mouse/rabbit IgG) .
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Subcellular fractionation approach: Separate nuclear and cytoplasmic fractions, then perform Western blot analysis to quantify ALDOA distribution between compartments.
For pathway interaction studies:
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Co-immunoprecipitation (Co-IP): Use 0.5-4.0 μg of ALDOA antibody per 1.0-3.0 mg of total protein lysate to pull down ALDOA and its binding partners .
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Pathway analysis: Assess the effects of ALDOA manipulation on signaling cascades, as exemplified by research showing ALDOA's influence on the AMPK/YAP pathway:
These approaches have revealed that ALDOA regulates YAP activity through the AMPK pathway in colorectal cancer cells, demonstrating how ALDOA antibodies can elucidate complex signaling networks .
What is the significance of ALDOA expression in cancer research?
ALDOA has emerged as an important factor in cancer biology, with antibody-based detection providing critical insights:
Prognostic significance:
Research utilizing ALDOA antibodies in immunohistochemical analyses has revealed that elevated ALDOA expression correlates with poor prognosis in colorectal cancer (CRC) patients with stage I-III disease . This indicates ALDOA's potential as a prognostic biomarker.
Functional roles in cancer progression:
Immunodetection of ALDOA has helped establish its involvement in:
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Tumor cell proliferation and migration
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Regulation of the YAP oncogenic pathway
Differential expression patterns:
Antibody-based analyses have demonstrated variable ALDOA expression across different cancer cell lines. For example, HT29, CaCo2, and DLD-1 colorectal cancer cells exhibit higher ALDOA expression compared to SW480 and SW620 cells . This heterogeneity may reflect different metabolic states or cancer progression stages.
How do serum anti-ALDOA antibody levels correlate with disease states?
Recent research has revealed interesting correlations between circulating anti-ALDOA antibodies and certain pathological conditions:
Cerebrovascular disease:
Studies utilizing amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) have demonstrated that patients with transient ischemic attack (TIA) or acute-phase cerebral infarction (aCI) exhibit significantly higher levels of anti-ALDOA antibodies (ALDOA-Ab) in their serum compared to healthy donors .
This suggests potential autoimmune responses involving ALDOA in cerebrovascular pathologies and highlights the possibility of using serum ALDOA-Ab levels as biomarkers for stroke risk or diagnosis .
Methodological considerations for serum antibody detection:
When investigating anti-ALDOA antibodies in serum or plasma:
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Select highly sensitive detection methods like AlphaLISA that can detect subtle differences in antibody levels
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Include appropriate cohorts (healthy controls and disease groups)
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Consider demographic factors that might influence antibody levels
These findings open new avenues for exploring ALDOA's role beyond its traditional metabolic functions and suggest potential applications in clinical diagnostics .
What emerging roles of ALDOA are being investigated in disease pathogenesis?
Beyond its well-established glycolytic function, antibody-based research has revealed ALDOA's involvement in several pathological processes:
Cancer metastasis and progression:
ALDOA appears to contribute to colorectal tumorigenesis and metastasis through regulation of the AMPK/YAP signaling axis. This has been demonstrated through antibody-based detection of pathway components following experimental manipulation of ALDOA expression .
Cerebrovascular disease:
The discovery of elevated anti-ALDOA antibodies in TIA and aCI patients suggests an unanticipated role in cerebrovascular pathologies, potentially involving autoimmune or inflammatory processes .
Metabolic regulation:
ALDOA has been shown to inhibit AMPK activation in various cell types, linking glycolytic metabolism to broader cellular signaling networks that influence cell growth, differentiation, and survival .
These emerging roles highlight the importance of continued investigation into ALDOA's multifaceted functions beyond glycolysis and suggest potential therapeutic targets for various diseases.
How can I minimize background and optimize signal-to-noise ratio with ALDOA antibodies?
Achieving optimal signal-to-noise ratio requires careful attention to several methodological aspects:
For Western Blot applications:
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Dilution optimization: While recommended ranges are 1:5000-1:50000, perform titration experiments to identify the optimal concentration for your specific sample type .
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Blocking optimization: Use freshly prepared blocking buffer (5% non-fat milk or BSA in TBST) and ensure adequate blocking time (1-2 hours at room temperature).
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Antibody quality: Consider premium antibodies like Picoband® that are specifically designed to provide strong signals with minimal background .
For IHC/IF applications:
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Antigen retrieval: Optimize based on tissue type, using either TE buffer (pH 9.0) or EDTA buffer (pH 8.0) .
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Blocking: Use 10% goat serum (or serum matching the secondary antibody host) to minimize non-specific binding .
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Antibody incubation: For IHC, incubate with primary antibody overnight at 4°C at optimized concentrations (typically 1:100-1:400 for IHC) .
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Detection system selection: For IHC, Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen has produced reliable results with minimal background .
These optimizations are essential for generating publication-quality data, particularly when studying ALDOA in complex samples like cancer tissues.
Why might I observe unexpected molecular weights when detecting ALDOA?
The expected molecular weight of ALDOA is 39 kDa (calculated), but observed weights may range from 35-40 kDa . This variation can result from several factors:
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Post-translational modifications: Phosphorylation, glycosylation, or other modifications may alter protein mobility in SDS-PAGE.
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Proteolytic processing: Sample handling or endogenous proteases may generate truncated forms.
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Alternative splicing: Different ALDOA isoforms may be expressed in different tissues.
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Sample preparation conditions: Denaturation methods and buffer compositions can affect protein migration patterns.
When troubleshooting unexpected band patterns:
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Compare your results with the validated molecular weight range (35-40 kDa for ALDOA)
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Include positive control samples from well-characterized sources (e.g., HEK-293, HeLa, or HepG2 cells)
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Consider running a gradient gel to better resolve proteins in this molecular weight range
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Verify antibody specificity using genetic approaches (knockdown/knockout samples)
Understanding these potential variations is crucial for accurate interpretation of experimental results when working with ALDOA antibodies.
Product Science Overview
Aldolase A, also known as fructose-bisphosphate aldolase, is a crucial enzyme in the glycolytic pathway. It catalyzes the reversible conversion of fructose-1,6-bisphosphate into glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) . This enzyme is encoded by the ALDOA gene in humans and plays a significant role in energy production within cells.
Aldolase A is a key enzyme in the fourth step of glycolysis and is also involved in gluconeogenesis . It is a member of the class I fructose-bisphosphate aldolase protein family and is expressed in various tissues, including muscle, liver, and brain . The enzyme’s activity is essential for maintaining cellular energy levels, especially in tissues with high energy demands.
The analysis of aldolase levels in the blood is used to assess muscle and liver function . Elevated aldolase levels may indicate muscle or liver damage, inflammation, or certain autoimmune diseases . Conversely, lower-than-normal levels might suggest a deficiency in aldolase production. This enzyme is also associated with Glycogen Storage Disease XII, an autosomal recessive disorder characterized by hemolytic anemia .
Mouse anti-human Aldolase-A antibodies are monoclonal antibodies developed to target and bind specifically to the human Aldolase-A protein. These antibodies are used in various scientific applications, including Western Blot, Immunocytochemistry, ELISA, Immunohistochemistry, and Immunoprecipitation . They are valuable tools for studying the expression and function of Aldolase-A in different biological contexts.
Mouse anti-human Aldolase-A antibodies have been used extensively in research to investigate the role of Aldolase-A in various diseases, including cancer and metabolic disorders . These antibodies help in identifying the presence and quantity of Aldolase-A in tissue samples, aiding in the understanding of its involvement in disease mechanisms.
