ACY1 Antibody

What is ACY1 and why is it important in research?

ACY1 (Aminoacylase-1) is a cytosolic, homodimeric, zinc-binding enzyme that catalyzes the hydrolysis of N-acetylated amino acids to acetate and free amino acids. It plays a significant role in the deacetylation process of N-acyl amino acids and functions in the catabolism and salvage of acylated amino acids . The gene is located on chromosome 3p21.1, a region reduced to homozygosity in small-cell lung cancer (SCLC) . Research interest in ACY1 has increased due to its differential expression in various cancer types, with reduced expression in SCLC and renal cell carcinoma but overexpression in colorectal cancer (CRC) .

The selection depends on your specific research needs:

Polyclonal ACY1 antibodies:

• Recognize multiple epitopes on the ACY1 protein

• Provide higher sensitivity due to binding multiple epitopes

• Examples include rabbit polyclonal antibodies (ab231332, ab189399)

• Ideal for initial detection and when signal amplification is needed

• Better tolerance of protein denaturation or modification

Monoclonal ACY1 antibodies:

• Recognize a single epitope on the ACY1 protein

• Provide higher specificity and reduced background

• Examples include mouse monoclonal antibodies (OTI1A12, OTI2F1)

• Ideal for distinguishing specific regions or isoforms

• More consistent lot-to-lot reproducibility

For novel research, using both types can provide complementary data and validation .

What controls should be included in ACY1 antibody experiments?

Proper controls are essential for result validation:

• Positive controls:

  • Recombinant human ACY1 protein

  • Pig kidney/liver lysates (known to express ACY1)

  • Human kidney tissue sections (high ACY1 expression)

  • K562 cells (human chronic myelogenous leukemia cell line)

• Negative controls:

  • Primary antibody omission control

  • Isotype control (matching IgG class)

  • SCLC cell lines (known to have reduced ACY1 expression)

  • Blocking peptide competition (pre-incubation with immunogen)

• Knockdown/knockout validation:

  • siRNA silencing of ACY1 in HCT116 cells has been demonstrated to effectively reduce ACY1 expression, providing a method for antibody specificity confirmation

How can I optimize ACY1 antibody concentration for my experiments?

Optimization is experiment-specific but follows general guidelines:

For Western Blot:

• Start with manufacturer's recommended dilution (typically 1:1,000-5,000)

• Perform titration experiments (e.g., 1:500, 1:1,000, 1:2,000, 1:5,000)

• Evaluate signal-to-noise ratio at each concentration

• Select concentration that provides clear band at expected molecular weight (46 kDa for ACY1)

For IHC/ICC:

• Begin with recommended concentration (10-20 μg/ml or 1:150-1:200 dilution)

• Test multiple concentrations on known positive tissue (kidney, liver)

• Assess specific staining versus background

• Optimize antigen retrieval method if needed

For Flow Cytometry:

• Start with 1:100 dilution as recommended

• Compare with isotype control

• Adjust to achieve clear population separation

What sample preparation methods work best for ACY1 detection?

Sample preparation varies by application:

For Western Blot:

• Effective lysis buffers: RIPA or NP-40 with protease inhibitors

• Recommended protein loading: 30-35 μg per lane

• Predicted band size: 46 kDa (main band), with possible additional bands at 40 kDa and 43 kDa in some cell lines

For IHC:

• Fixation: Formalin-fixed, paraffin-embedded (FFPE) tissues

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Detection systems: DAB staining provides good results with ACY1 antibodies

For ICC/IF:

• Fixation: Paraformaldehyde fixation (4%)

• Permeabilization: 0.1% Triton X-100

• Blocking: 1-5% BSA or normal serum

How can ACY1 antibodies be utilized in cancer research?

ACY1 antibodies are valuable tools in cancer research due to the differential expression of ACY1 across tumor types:

• Colorectal Cancer (CRC):

  • ACY1 is overexpressed in CRC and associated with advanced TNM stage

  • Strong ACY1 expression correlates with lymph node metastasis, positive vascular invasion, and shorter cancer-specific survival

  • ACY1 knockdown inhibits cell proliferation and induces apoptosis in HCT116 cells

  • Immunohistochemical analysis with ACY1 antibodies can help evaluate ACY1 as a prognostic marker

• Small Cell Lung Cancer (SCLC):

  • ACY1 expression is reduced or undetectable in SCLC cell lines and tumors

  • Located on chromosome 3p21.1, a region reduced to homozygosity in SCLC

  • ACY1 antibodies can be used to study loss of expression in comparison to normal lung tissue

• Experimental approaches:

  • Tissue microarray analysis with ACY1 antibodies

  • Correlation of staining intensity with clinicopathological parameters

  • Combined approaches with proliferation markers

  • Analysis of downstream signaling pathways affected by ACY1 expression

What methodological challenges exist in ACY1 detection and quantification?

Researchers should be aware of several technical challenges:

• Isoform detection:

  • Multiple predicted bands (40 kDa, 43 kDa, 46 kDa) may be observed in Western blot

  • Different antibodies may preferentially detect certain isoforms

  • Read-through transcription exists between ACY1 and the upstream ABHD14A gene

• Quantification methods:

  • For IHC: Standardized scoring systems (e.g., H-score, percentage of positive cells)

  • For WB: Normalization to housekeeping proteins (β-actin, GAPDH)

  • For IF: Mean fluorescence intensity measurements with appropriate software

• Cross-reactivity considerations:

  • A related pseudogene has been identified on chromosome 18

  • Verify antibody specificity across species (human, mouse, rat, dog)

  • Consider using multiple antibodies targeting different epitopes

• Background reduction strategies:

  • Optimized blocking (5% BSA or milk)

  • Careful antibody titration

  • Extended washing steps

  • Use of monoclonal antibodies for higher specificity

How can ACY1 antibodies be employed in functional studies?

Beyond detection, ACY1 antibodies can facilitate functional investigations:

• Protein-protein interaction studies:

  • Co-immunoprecipitation using ACY1 antibodies

  • Proximity ligation assays to detect in situ interactions

  • Immunofluorescence co-localization studies

• Enzyme activity correlation:

  • Combine ACY1 antibody detection with enzymatic activity assays

  • Correlate protein levels with functional deacetylation activity

  • Investigate zinc-binding properties using specialized assays alongside antibody detection

• Knockdown/overexpression validation:

  • Confirm siRNA-mediated knockdown efficiency by Western blot

  • Validate overexpression constructs

  • Track changes in subcellular localization after manipulation

• Tissue-specific expression analysis:

  • Multi-tissue Western blot panels

  • Immunohistochemical tissue microarrays

  • Correlation with transcriptomic data

What are common issues with ACY1 antibodies and their solutions?

How can I verify the specificity of my ACY1 antibody?

Verification strategies include:

• Molecular weight confirmation:

  • ACY1 has a predicted molecular weight of 46 kDa

  • Use recombinant ACY1 protein as positive control

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Specific binding should be blocked

• Genetic approaches:

  • siRNA knockdown (as demonstrated in HCT116 cells)

  • CRISPR/Cas9 knockout validation

  • Compare antibody reactivity before and after genetic manipulation

• Multi-antibody comparison:

  • Use antibodies raised against different epitopes

  • Consistent patterns support specificity

• Tissue/cell expression pattern:

  • Known high expression: kidney, liver

  • Low expression: SCLC cell lines

What alternative approaches can complement ACY1 antibody studies?

To strengthen research findings, consider complementary techniques:

• Transcriptomic analysis:

  • RT-qPCR for ACY1 mRNA levels

  • RNA-seq to identify correlating genes

  • Correlation between protein and mRNA levels

• Mass spectrometry:

  • Protein identification and quantification

  • Post-translational modification analysis

  • Interaction partner identification

• Functional assays:

  • Enzymatic activity assays for ACY1

  • Metabolomic analysis of N-acetylated amino acids

  • Cell proliferation and apoptosis assays following ACY1 manipulation

• In silico analysis:

  • Structure prediction and modeling

  • Analysis of mutation effects on protein structure

  • Gene expression correlation analysis across databases

These complementary approaches can provide a more comprehensive understanding of ACY1 biology when used alongside antibody-based methods.