ACY1 Antibody

What is ACY1 and what is its primary function in cellular metabolism?

ACY1 (Aminoacylase 1) is a cytosolic enzyme that catalyzes the hydrolysis of N-acetylated amino acids to acetate and free amino acids during intracellular protein degradation . Beyond its catalytic role in aminoacylation, ACY1 has important metabolic functions, interacting with proteins such as acetyl-CoA synthetase to convert excess acyl-CoAs into free fatty acids and CoA, thereby preventing the accumulation of toxic metabolites . ACY1 is widely expressed throughout various organs, with particularly high abundance in epithelial cells of the human digestive tract .

Which ACY1 antibody clones are most validated for research applications?

Several validated clones are available for ACY1 research, including:

• Mouse monoclonal antibody clone OTI1A12, which is available carrier-free (BSA/glycerol-free)

• Mouse monoclonal antibody clone 1F10-H10-G9, which is validated for ELISA and Western blotting applications

• Rabbit polyclonal antibody (ab231332), which has been validated for Western blot and immunohistochemistry applications in human and pig samples

The choice of clone depends on your specific experimental needs and target species. For human ACY1 research, all three options have demonstrated efficacy, though with different application strengths.

What are the recommended applications for ACY1 antibodies in research?

ACY1 antibodies have been validated for multiple research applications:

For optimal results, researchers should perform preliminary dilution tests for their specific experimental conditions and sample types .

How should I optimize ACY1 antibody-based immunohistochemistry protocols for formalin-fixed tissues?

For optimal ACY1 detection in formalin-fixed, paraffin-embedded (FFPE) tissues:

• Use heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) for 20 minutes

• Block endogenous peroxidase with 3% hydrogen peroxide

• Apply ACY1 antibody at 20 μg/ml concentration for human tissues

• Incubate overnight at 4°C

• Use DAB (3,3'-diaminobenzidine) for visualization

This protocol has been validated for human liver tissue and can be adapted for other tissue types. When analyzing colorectal cancer specimens, consider that ACY1 expression is particularly abundant in epithelial cells rather than stromal cells , which may influence interpretation of staining patterns.

What are the recommended protocols for detecting ACY1 autoantibodies in serum samples?

For detecting ACY1 autoantibodies in serum samples using ELISA:

• Coat 96-well microplates with ACY1 protein at 4 μg/mL in carbonate/bicarbonate buffer (pH 9.6)

• Incubate at 37°C for 2 hours, then overnight at 4°C

• Wash with PBS containing 0.05% Tween-20

• Block with 10% newborn bovine serum (NBS) in PBS

• Dilute patient serum 1:100 in 10% NBS and incubate for 1 hour at 37°C

• Use HRP-conjugated anti-human IgG at 1:15,000 dilution in 20% NBS

• Develop with TMB substrate and read at 450nm

This protocol has been successfully used to detect ACY1 autoantibodies in patients with liver fibrosis, with results indicating potential diagnostic value .

What are the predicted band sizes for ACY1 detection in Western blotting experiments?

When performing Western blot analysis for ACY1:

• The predicted band sizes are 40 kDa, 43 kDa, and 46 kDa

• The predominant band appears at approximately 46 kDa

• Multiple bands may represent different isoforms or post-translational modifications of ACY1

To confirm antibody specificity, use recombinant human ACY1 protein as a positive control . Non-specific bands may appear in complex tissue lysates, so appropriate controls and blocking optimizations are essential for accurate interpretation.

How does ACY1 expression differ between colorectal cancer and normal tissues?

Research has demonstrated significant differences in ACY1 expression between colorectal cancer and normal tissues:

• ACY1 mRNA levels are significantly increased in colorectal tumor tissue compared to adjacent normal tissue

• ACY1 protein levels are notably elevated in colorectal cancer specimens

• Plasma ACY1 concentration is higher in patients with colorectal cancer compared to healthy controls

• ACY1 mRNA expression positively correlates with tumor stage, with higher expression in more advanced stages

These findings contradict observations in small cell lung cancer and liver cancer, where ACY1 expression is reduced or undetectable . This tissue-specific expression pattern suggests distinct regulatory mechanisms and functions of ACY1 in different cancer types.

What signaling pathways are affected by ACY1 in cancer cell proliferation?

ACY1 influences cancer cell proliferation through several interconnected signaling pathways:

• ERK1/2 pathway: Knockdown of ACY1 in colorectal cancer cells leads to decreased ERK1/2 phosphorylation

• TGF-β1 signaling: ACY1 silencing results in reduced TGF-β1 expression

• Anti-apoptotic mechanisms: ACY1 interacts with Sphingosine kinase type 1 (SphK1), an anti-apoptosis protein that enhances its anti-apoptotic effect

This signaling cascade suggests that ACY1 promotes tumor progression by activating the ERK pathway, which increases TGF-β1 expression, ultimately leading to enhanced proliferation and reduced apoptosis in cancer cells . These relationships provide potential therapeutic targets for colorectal cancer treatment strategies.

Can serum ACY1 levels be used as a diagnostic biomarker for colorectal cancer?

Evidence suggests that serum ACY1 could serve as a potential diagnostic biomarker for colorectal cancer:

• Serum ACY1 protein levels are significantly elevated in patients with colorectal cancer compared to healthy controls

• A positive correlation exists between serum ACY1 concentration and colorectal cancer progression

• The increased serum levels likely reflect increased expression in tumors

How should I design experiments to investigate the functional role of ACY1 in cancer cell lines?

To investigate ACY1's functional role in cancer:

• Expression analysis:

  • Compare ACY1 mRNA and protein levels between normal and cancer cell lines

  • Correlate expression with proliferative capacity and invasiveness

• Loss-of-function studies:

  • Use siRNA silencing to knockdown ACY1 expression as demonstrated in HT-29 colorectal cancer cells

  • Assess effects on:

    • Cell proliferation using CCK-8 assays

    • Cell migration using scratch assays

    • Apoptosis rates

• Signaling pathway analysis:

  • Monitor changes in key signaling molecules (ERK1/2 phosphorylation, TGF-β1 expression)

  • Use pathway inhibitors to confirm mechanism

• In vivo validation:

  • Develop xenograft models with ACY1-knockdown cells

  • Compare tumor growth rates and metastatic potential

This comprehensive approach has successfully demonstrated that ACY1 knockdown inhibits proliferation and increases apoptosis in colorectal cancer cells , providing a framework for investigating its role in other cancer types.

What experimental controls should be included when using ACY1 antibodies for immunohistochemistry in cancer tissue microarrays?

For robust immunohistochemical analysis of ACY1 in cancer tissue microarrays:

• Positive tissue controls:

  • Human liver tissue (known to express ACY1)

  • Normal colonic epithelium (abundant ACY1 expression)

• Negative controls:

  • Primary antibody omission

  • Isotype control antibody

  • Tissues known to lack ACY1 expression

• Antibody validation controls:

  • Pre-absorption with recombinant ACY1 protein

  • Comparison with a second validated ACY1 antibody

• Technical controls:

  • Range of antibody concentrations (titration)

  • Different antigen retrieval methods

  • Multiple tissue samples per patient

• Analysis controls:

  • Blinded scoring by multiple pathologists

  • Quantitative image analysis with standardized parameters

These controls enable reliable interpretation of ACY1 expression patterns in tissue microarrays composed of multiple cancer cases, as demonstrated in studies examining 120 colorectal cancer specimens .

How do I resolve contradictory findings regarding ACY1 expression across different cancer types?

Research shows contradictory findings regarding ACY1 expression in different cancers:

• Decreased expression observed in:

  • Small cell lung cancer (SCLC) - undetectable levels

  • Liver cancer - reduced expression

• Increased expression observed in:

  • Colorectal cancer - significantly elevated levels

To resolve these contradictions:

• Standardize detection methods:

  • Use multiple antibody clones with different epitope recognition

  • Employ both mRNA and protein detection methods

  • Quantify expression using absolute quantification methods

• Consider tissue context:

  • Account for baseline expression in corresponding normal tissues

  • Analyze expression in epithelial vs. stromal compartments

• Examine cancer subtypes:

  • Stratify analyses by molecular and histological subtypes

  • Consider stage-specific expression patterns

• Investigate regulatory mechanisms:

  • Analyze promoter methylation status

  • Examine microRNA regulation

  • Assess copy number variations

These tissue-specific differences likely reflect distinct regulatory mechanisms and functional roles of ACY1 in different cancer types, highlighting the importance of context-specific interpretation of ACY1 expression data .

What technical challenges might I encounter when measuring ACY1 activity versus protein levels in experimental systems?

Researchers face several challenges when comparing ACY1 enzymatic activity versus protein levels:

• Post-translational modifications:

  • ACY1 function can be regulated by modifications not reflected in protein quantity

  • Activity assays may better represent functional status than expression levels

• Protein stability considerations:

  • ACY1 has multiple isoforms (40, 43, and 46 kDa)

  • Sample preparation methods may differentially affect protein stability

• Assay specificity issues:

  • Enzymatic assays may detect activity from related aminoacylases

  • Antibodies may cross-react with structurally similar proteins

• Enzyme kinetics variability:

  • Environmental factors (pH, temperature, ion concentration) affect activity measurements

  • Substrate specificity varies across experimental conditions

• Technical recommendations:

  • Perform parallel protein quantification and activity assays

  • Include recombinant ACY1 standards with known activity

  • Use multiple complementary detection methods

Addressing these challenges enables more reliable correlation between ACY1 expression and its functional significance in experimental systems, particularly important when investigating its role in disease processes.

What are promising future approaches for targeting ACY1 in cancer therapeutics?

Based on current understanding of ACY1's role in cancer, several promising therapeutic approaches warrant investigation:

• Small molecule inhibitors:

  • Develop specific ACY1 enzymatic activity inhibitors

  • Target ACY1-protein interactions (e.g., with SphK1)

• Downstream pathway modulation:

  • Combined inhibition of ACY1 and ERK signaling

  • TGF-β1 pathway antagonists in ACY1-overexpressing tumors

• RNA-based therapeutics:

  • siRNA delivery systems for ACY1 knockdown in tumors

  • CRISPR-Cas9 approaches for genetic modification

• Immunological approaches:

  • ACY1 autoantibody-based diagnostics

  • Targeting ACY1 in immunotherapy protocols

• Biomarker development:

  • Serum ACY1 for early detection and monitoring

  • Prediction of treatment response based on ACY1 expression levels

These approaches leverage the observation that ACY1 knockdown inhibits proliferation and increases apoptosis in colorectal cancer cells , suggesting therapeutic potential for ACY1-targeting strategies.

How might technological advances in antibody development improve future ACY1 research?

Emerging antibody technologies will enhance ACY1 research capabilities:

• Single-domain antibodies (nanobodies):

  • Higher specificity for ACY1 epitopes

  • Better penetration in tissue sections and live cell imaging

  • Enhanced stability for challenging applications

• Recombinant antibody engineering:

  • Humanized ACY1 antibodies for therapeutic applications

  • Bispecific antibodies targeting ACY1 and complementary cancer markers

  • Antibody fragments optimized for specific research applications

• Advanced detection systems:

  • Quantum dot-conjugated ACY1 antibodies for multiplexed imaging

  • Mass cytometry (CyTOF) compatible ACY1 antibodies

  • CODEX and other spatial proteomics technologies

• Functional antibodies:

  • Intrabodies for tracking and modulating ACY1 in living cells

  • Antibody-drug conjugates for targeted therapy

  • Activating or inhibiting antibodies affecting ACY1 function

These technological advances will facilitate more comprehensive characterization of ACY1's role in normal physiology and disease, potentially enabling translation of basic research findings into clinical applications.