ACLY Monoclonal Antibody

What is ATP citrate lyase (ACLY) and why is it significant in research?

ATP citrate lyase (ACLY) is a critical metabolic enzyme that catalyzes the conversion of citrate to acetyl-CoA, providing essential building blocks for fatty acid and cholesterol biosynthesis. This enzyme plays a pivotal role in cellular lipid metabolism and has emerged as an important research target in multiple disease contexts. ACLY has been identified as a novel host factor required for efficient replication of SARS-CoV-2, including wild-type and variant strains such as Omicron . Additionally, research has demonstrated ACLY's crucial function in lipogenesis and its potential interaction with the immune microenvironment in hepatocellular carcinoma (HCC) . The central position of ACLY in metabolic pathways makes it an attractive target for studying disease mechanisms and potential therapeutic interventions.

What applications are validated for ACLY monoclonal antibodies?

ACLY monoclonal antibodies have been validated for multiple experimental applications, providing researchers with versatile tools for investigating this enzyme. According to validated data, high-quality ACLY monoclonal antibodies can be effectively used in:

• Western Blot (WB)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF)

• Immunocytochemistry (ICC)

• Flow Cytometry

For example, the monoclonal antibody (clone 5I2) has been specifically tested and validated across all these applications with human, mouse, and rat samples . This multi-application validation ensures researchers can study ACLY expression and localization using various complementary techniques.

What species reactivity should researchers expect from ACLY monoclonal antibodies?

When selecting an ACLY monoclonal antibody, species reactivity is a crucial consideration for experimental design. Many commercially available ACLY monoclonal antibodies, such as the Picoband® monoclonal antibody (clone 5I2), demonstrate cross-reactivity with human, mouse, and rat ACLY . This cross-species reactivity is particularly valuable for translational research that involves both human samples and animal models, allowing for consistent detection methodology across different experimental systems.

How does ACLY contribute to SARS-CoV-2 replication mechanisms?

Recent metabolomic analyses combined with in vitro and in vivo investigations have identified ACLY as a critical host factor required for efficient SARS-CoV-2 replication. Through detailed metabolome studies of COVID-19 patients, researchers determined that ACLY plays an essential role in supporting viral replication . The mechanism appears to involve ACLY's function in lipid biosynthesis, as SARS-CoV-2 requires substantial lipid resources for various steps in its life cycle, including membrane formation for replication complexes, virion assembly, and budding.

Importantly, ACLY inhibition effectively disturbs viral replication by reducing the availability of lipids crucial for these processes. This finding extends across multiple SARS-CoV-2 variants, including the Omicron variant, suggesting ACLY represents a potential host-directed therapeutic target that may remain effective despite viral mutations that affect other treatment approaches .

What role does ACLY play in cancer metabolism and progression?

ACLY has emerged as a significant metabolic regulator in cancer, particularly in hepatocellular carcinoma (HCC). Transcriptomic and metabolomic approaches have identified ACLY as a novel signature in HCC with portal vein tumor thrombosis (PVTT) . Research demonstrates that ACLY oncogenic activation triggers metabolism adaptations in both fatty acid and cholesterol biosynthesis pathways, supporting the high energy and biomass requirements of rapidly proliferating cancer cells.

Beyond its direct metabolic functions, ACLY exhibits important interactions with the tumor immune microenvironment. Studies have revealed correlations between ACLY expression and CD276 (B7-H3), a promising target in immune checkpoint blockade therapy . This relationship suggests that ACLY may influence tumor immune evasion mechanisms. Furthermore, combination analyses of ACLY expression, CD276 levels, and immune infiltration status have demonstrated significant prognostic value for risk stratification in HCC patients .

How should researchers design experiments to investigate ACLY inhibition effects?

When designing experiments to investigate ACLY inhibition, researchers should consider a multi-faceted approach that captures both direct metabolic effects and downstream consequences on disease-specific processes. Based on current research methodologies, a comprehensive experimental design would include:

• Metabolic profiling: Ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) to quantify changes in lipid metabolites before and after ACLY inhibition .

• Transcriptomic analysis: RNA-sequencing to identify differentially expressed genes and enriched pathways following ACLY inhibition, focusing on lipid metabolism genes and disease-specific pathways.

• Functional assays: For viral studies, viral replication assays with quantification of viral load and infectious particle production . For cancer studies, proliferation, migration, and invasion assays.

• Immunological assessment: Evaluation of immune cell infiltration and function, particularly in cancer models, using techniques such as CIBERSORTx for immune deconvolution .

• In vivo validation: Animal models to confirm in vitro findings and evaluate potential therapeutic applications.

This multi-modal approach allows researchers to comprehensively characterize how ACLY inhibition affects not only metabolic parameters but also disease-specific outcomes.

What protocol is recommended for immunohistochemistry with ACLY antibodies?

For successful immunohistochemical detection of ACLY in tissue samples, researchers should follow a validated protocol that ensures specific signal with minimal background. Based on published methodologies, the following IHC-P protocol has been validated for ACLY detection:

IHC Protocol for ACLY Detection:

• Sample preparation: Use paraffin-embedded tissue sections (e.g., pancreas tissue from mouse or rat models has been validated).

• Antigen retrieval: Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0, epitope retrieval solution).

• Blocking: Block the tissue section with 10% goat serum to reduce non-specific binding.

• Primary antibody incubation: Incubate with 1μg/ml mouse anti-ATP citrate lyase antibody overnight at 4°C.

• Secondary antibody: Apply biotinylated goat anti-mouse IgG as secondary antibody with 30-minute incubation at 37°C.

• Detection system: Develop using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.

• Counterstaining: Apply appropriate counterstain based on experimental needs .

This protocol has successfully demonstrated ACLY expression in mouse and rat pancreas tissues, with clear cytoplasmic staining patterns consistent with ACLY's known cellular localization .

What are the critical parameters for flow cytometry analysis of ACLY expression?

Flow cytometry analysis of ACLY expression requires careful attention to cell preparation and antibody application due to ACLY's intracellular localization. A validated protocol includes the following key steps:

Flow Cytometry Protocol for ACLY Detection:

• Cell fixation: Fix cells with 4% paraformaldehyde to preserve cellular architecture.

• Permeabilization: Apply appropriate permeabilization buffer to allow antibody access to intracellular antigens.

• Blocking: Block with 10% normal goat serum to reduce non-specific binding.

• Primary antibody: Incubate with mouse anti-ATP citrate lyase antibody (1μg/1×10^6 cells) for 30 minutes at 20°C.

• Secondary antibody: Apply DyLight®488 conjugated goat anti-mouse IgG for 30 minutes at 20°C.

• Controls: Include both isotype control antibody (mouse IgG, 1μg/1×10^6 cells) and unlabelled sample as controls .

This approach has been validated with A549 cells, showing clear detection of ACLY with appropriate controls to confirm specificity . The inclusion of proper controls is critical for accurate interpretation of flow cytometry results, particularly for intracellular targets like ACLY.

How can researchers validate ACLY antibody specificity for their experimental system?

Antibody validation is a critical step in ensuring experimental reliability and reproducibility. For ACLY studies, comprehensive validation should include:

• Multi-application testing: Confirm antibody performance across different applications relevant to your research (WB, IHC, IF, Flow).

• Positive and negative controls: Include tissues/cells known to express high levels of ACLY (positive control) and those with minimal expression (negative control).

• Knockdown/knockout validation: Where possible, validate specificity using ACLY knockdown or knockout samples to confirm signal reduction.

• Cross-reactivity assessment: For studies involving multiple species, verify species cross-reactivity with appropriate control samples.

• Batch-to-batch consistency: When using the antibody for long-term studies, verify consistency between antibody lots.

• Signal-to-noise optimization: Titrate antibody concentration to determine optimal working dilution that maximizes specific signal while minimizing background .

Researchers should document validation results thoroughly to support the reliability of their findings and facilitate reproducibility by other laboratories.

What are common challenges in ACLY antibody applications and how can they be addressed?

When working with ACLY monoclonal antibodies, researchers may encounter several technical challenges. Here are common issues and recommended solutions:

For Western Blot:

• Weak signal: Try increasing antibody concentration, extending incubation time, or enhancing detection methods.

• Multiple bands: Optimize blocking conditions, increase washing stringency, or verify sample preparation to prevent protein degradation.

For Immunohistochemistry:

• High background: Adjust blocking conditions, optimize antibody dilution, or modify antigen retrieval method.

• Weak or absent staining: Consider different antigen retrieval methods, increase antibody concentration, or extend incubation times.

For Flow Cytometry:

• Poor separation between positive and negative populations: Optimize permeabilization conditions, adjust antibody concentration, or improve blocking strategy.

• High autofluorescence: Use appropriate compensation controls and consider fluorophores with spectral properties distinct from cellular autofluorescence .

How can ACLY antibodies be utilized in multiplexed immunofluorescence studies?

For researchers interested in studying ACLY in relation to other proteins or cellular structures, multiplexed immunofluorescence offers powerful capabilities. When incorporating ACLY antibodies into multiplexed studies:

• Antibody compatibility: Select primary antibodies raised in different host species to allow simultaneous detection without cross-reactivity.

• Fluorophore selection: Choose fluorophores with minimal spectral overlap to allow clear discrimination between signals.

• Sequential staining: For complex panels, consider sequential staining protocols with appropriate blocking between rounds.

• Colocalization analysis: Use appropriate imaging software for quantitative colocalization analysis between ACLY and other proteins of interest.

• Controls: Include single-stained controls and fluorescence-minus-one controls to accurately set thresholds and compensation.

For example, MCF7 cells have been successfully stained for ACLY using mouse anti-ATP citrate lyase antibody with DyLight®488 Conjugated Goat Anti-Mouse IgG as secondary antibody, counterstained with DAPI for nuclear visualization . This approach can be expanded to include additional markers relevant to metabolic pathways or disease processes.

How should researchers integrate ACLY expression data with other -omics datasets?

Modern research increasingly requires integration of multiple data types for comprehensive understanding of biological processes. For ACLY research, effective data integration strategies include:

• Transcriptomic correlation: Correlate ACLY protein expression (from antibody-based studies) with mRNA expression data to identify potential post-transcriptional regulation.

• Metabolomic integration: Link ACLY expression levels with metabolomic profiles, particularly focusing on lipid metabolites that are downstream of ACLY activity.

• Multi-omics analysis platforms: Utilize computational platforms that facilitate integration of protein expression, transcriptomic, and metabolomic data.

• Pathway analysis: Employ pathway enrichment tools to contextualize ACLY expression within broader metabolic and signaling networks.

Research has demonstrated the value of this integrative approach, with studies combining transcriptomic data (from GEO and TCGA databases) with metabolomic analyses to identify ACLY's role in cancer progression and its relationship with immune parameters . Similar integrative approaches can be applied to understanding ACLY's role in viral infection and other disease contexts.

What emerging applications of ACLY antibodies show promise for translational research?

As understanding of ACLY's role in disease processes expands, several emerging applications of ACLY antibodies hold particular promise for translational research:

• Diagnostic biomarker development: ACLY expression analysis may serve as a prognostic or predictive biomarker in cancer, particularly when combined with immune markers like CD276 .

• Therapeutic response monitoring: ACLY antibodies could be utilized to monitor changes in ACLY expression or activity during treatment with metabolism-targeting therapies.

• Patient stratification: Immunohistochemical analysis of ACLY in patient samples may help identify individuals likely to benefit from metabolism-targeted interventions.

• Imaging probe development: Derivatized ACLY antibodies could potentially be developed into imaging probes for non-invasive monitoring of metabolic activity in vivo.

• Combination therapy assessment: ACLY antibodies may help evaluate synergistic effects between metabolic inhibitors and other therapeutic approaches, such as immune checkpoint inhibitors in cancer .

These applications reflect the growing recognition of ACLY as not merely a metabolic enzyme but a key regulator at the intersection of metabolism, immunity, and disease progression.

What methodological advances are needed to enhance ACLY detection in challenging samples?

Despite substantial progress in ACLY antibody applications, several methodological challenges remain, particularly for certain sample types or experimental contexts:

• Low abundance detection: Development of signal amplification strategies for detecting low ACLY expression in minimally invasive samples or single-cell applications.

• Phosphorylation-specific detection: Generation and validation of antibodies specific to phosphorylated ACLY, which represents the active form of the enzyme.

• Tissue microenvironment analysis: Improved methods for spatial analysis of ACLY expression within heterogeneous tissues, potentially through multiplex imaging technologies.

• Live-cell ACLY activity monitoring: Development of antibody-based biosensors that could monitor ACLY activity in living cells without fixation requirements.

• Automation and standardization: Establishment of automated, standardized protocols for ACLY detection across different sample types to enhance reproducibility and facilitate clinical translation.

Addressing these methodological challenges will expand the utility of ACLY antibodies in both basic and translational research contexts, potentially accelerating the development of ACLY-targeted therapeutic approaches.