ACAT2 Antibody

What is ACAT2 and why are antibodies against it important in research?

ACAT2 is a subtype of Acetyl-coenzyme A acetyltransferase (ACAT), a membrane-bound enzyme that plays important roles in lipid metabolism. It participates in isoleucine degradation, ketolysis, ketogenesis, and fatty acid oxidation . ACAT2 antibodies are crucial research tools that enable the detection, quantification, and localization of ACAT2 in various experimental contexts, particularly in cancer research where ACAT2 has been identified as a potential biomarker and therapeutic target. Recent studies have shown that ACAT2 is significantly upregulated in gastric cancer tissues compared to normal counterparts, and high expression correlates with poor prognosis .

What are the recommended methods for detecting ACAT2 expression in tissue samples?

Several methodological approaches are effective for detecting ACAT2 expression:

• Immunohistochemistry (IHC): For tissue samples, IHC can effectively detect ACAT2 with optimal antibody dilution of 1:300 (for antibodies like ab131215, Abcam). The protocol involves:

  • Sectioning paraffin-embedded tissues at 4 μm thickness

  • Baking at 65°C for 2 hours

  • Deparaffinizing in xylene and rehydrating through graded alcohol

  • Placing in 3% hydrogen peroxide

  • Heating in 1x sodium citrate antigen retrieval buffer (pH 6.0) using a pressure cooker

  • Blocking with 5% bovine serum albumin

  • Incubating with primary antibody overnight at 4°C

  • Incubating with secondary antibody (e.g., MaxVision-HRP) for 1 hour

  • Visualizing with diaminobenzidine and counterstaining with hematoxylin

• Western Blotting: For cell lines, western blotting can be performed using:

  • RIPA lysis buffer with 1 μm phenylmethanesulfonyl fluoride

  • 10% sodium dodecyl sulfate–polyacrylamide gel

  • Polyvinylidene fluoride membrane (0.45 μm)

  • Primary antibody dilution of 1:5000 (for antibodies like ab131215, Abcam)

  • Horseradish peroxidase-conjugated secondary antibodies at 1:3000 dilution

• Quantitative Real-Time PCR: For mRNA expression:

  • RNA extraction using TRIzol reagent

  • cDNA synthesis

  • qPCR using primers specific to ACAT2

  • Recommended primer sequences: 5'-GCCTTCCATTATGGGAATAGGA-3' and 5'-GACCTTCTCTGGGTTTAATCCA-3'

How should I score and interpret ACAT2 immunohistochemistry results?

ACAT2 immunostaining evaluation requires careful scoring and interpretation:

• Localization: ACAT2 staining is typically observed in both the cytoplasm and nucleus of cells .

• Scoring Method: A comprehensive scoring system involves:

  • Selecting five high-power fields randomly in each specimen

  • Examining cytoplasm staining

  • Calculating immune score = percentage of positive cells × staining intensity

  • Percentage scoring: 0 (≤5%), 1 (6−25%), 2 (26−50%), 3 (51−75%), 4 (76−100%)

  • Intensity scoring: 0 (negative), 1 (weak), 2 (moderate), 3 (strong)

  • Final immune score range: 0-12

• Expression Classification:

  • Low expression: Immune scores of 0–4

  • High expression: Immune scores of 5–12

This standardized approach facilitates reliable comparison across different studies and samples.

How can ACAT2 antibodies be utilized to investigate its role in cancer progression mechanisms?

ACAT2 antibodies are instrumental in elucidating the molecular mechanisms of ACAT2-mediated cancer progression:

• YAP1/TAZ-TEAD1 Signaling Pathway Analysis:

  • Co-immunoprecipitation with ACAT2 antibodies can identify interactions with YAP1

  • Immunoblotting for YAP1 after ACAT2 knockdown reveals changes in protein degradation

  • Immunofluorescence co-staining of ACAT2 and YAP1 can demonstrate subcellular co-localization

• SETD7 Expression Regulation:

  • Chromatin immunoprecipitation using ACAT2 antibodies can identify direct binding to the SETD7 promoter

  • Western blotting for SETD7 after ACAT2 manipulation demonstrates regulatory relationships

  • Research has shown that ACAT2 depletion significantly reduced SETD7 transcription, and ACAT2's pro-tumoral functions were largely dependent on SETD7

• EMT Process Evaluation:

  • Multiplex immunostaining with ACAT2 and EMT markers (E-cadherin, N-cadherin, vimentin, SMA, snail2)

  • Studies have shown that ACAT2 knockdown upregulates E-cadherin and downregulates N-cadherin, vimentin, SMA, and snail2, indicating ACAT2's role in promoting EMT

• Cell Cycle Regulation:

  • Flow cytometry combined with ACAT2 antibody detection can analyze cell cycle distribution

  • Western blotting for p21 (CDKN1A) demonstrates ACAT2's impact on cell cycle regulators

  • ACAT2 knockdown has been shown to block the cell cycle in G1/S phase and increase p21 expression

What methodological approaches are recommended for studying ACAT2 in chemotherapy resistance?

Several methodological approaches can be employed:

How can contradictory ACAT2 expression data across different cancer types be resolved?

Resolving contradictory data requires systematic approaches:

• Standardization of Detection Methods:

  • Use consistent antibody clones and dilutions across studies

  • Implement uniform scoring systems for immunohistochemistry

  • Include validated positive and negative controls

• Context-Specific Analysis:

  • Evaluate ACAT2 function in specific cellular contexts

  • Consider tissue-specific roles and metabolic environments

  • Research has shown that ACAT2 can have different effects in different cancer types

• Pathway Intersection Analysis:

  • Examine ACAT2's interaction with different signaling networks in various cancer types

  • Map tissue-specific downstream targets

  • In gastric cancer, ACAT2 interacts with the SETD7-YAP1 axis, which might differ in other cancers

• Meta-analysis Approach:

  • Systematically combine data from multiple studies

  • Account for methodological variations

  • Analyze subgroup differences based on cancer type, stage, and treatment history

What controls are essential when designing experiments with ACAT2 antibodies?

Robust experimental design requires comprehensive controls:

• For Western Blotting:

  • Positive control: Cell lines known to express high levels of ACAT2 (e.g., HGC-27, NCI-N87 for gastric cancer; A2780/DDP for ovarian cancer)

  • Negative control: Cell lines with minimal ACAT2 expression or ACAT2 knockdown samples

  • Loading control: GAPDH (recommended dilution 1:50000) or other housekeeping proteins

  • Molecular weight marker to confirm the expected size of ACAT2 (approximately 41 kDa)

• For Immunohistochemistry:

  • Positive tissue control: Gastric cancer or ovarian cancer tissues with confirmed high ACAT2 expression

  • Negative tissue control: Normal gastric or ovarian tissues

  • Technical negative control: Primary antibody omission

  • Isotype control: Non-specific antibody of the same isotype and concentration

• For Gene Expression Studies:

  • Endogenous control: GAPDH or other stable reference genes

  • No-template controls for PCR

  • Standard curves for absolute quantification

  • Statistical validation across biological replicates

What is the optimal protocol for ACAT2 knockdown validation?

A comprehensive validation protocol includes:

• Design Phase:

  • Select target sequences for shRNA design (multiple sequences recommended)

  • Include non-targeting control shRNA

  • Consider inducible knockdown systems for temporal control

• Knockdown Implementation:

  • Transduce lentiviral shRNA constructs into target cell lines

  • Select stable knockdown cell lines using appropriate antibiotics

  • Optimize MOI (multiplicity of infection) for efficient transduction

• Validation Methods:

  • mRNA Level: qRT-PCR with ACAT2-specific primers

  • Protein Level: Western blotting with ACAT2 antibody (1:5000 dilution recommended)

  • Functional Validation: Proliferation assays (CCK-8), colony formation assays, migration assays (wound healing), invasion assays (transwell), and cell cycle analysis by flow cytometry

• Phenotypic Confirmation:

  • In vivo tumor growth assessment in immunocompromised mice

  • Ki-67 staining of tumor sections to confirm proliferation changes

  • Studies have shown that ACAT2 depletion significantly reduced tumor growth and proliferation in xenograft models

What methods should be used to investigate the relationship between ACAT2 and ubiquitination pathways?

To investigate ACAT2's role in ubiquitination:

• Co-immunoprecipitation Assays:

  • Immunoprecipitate YAP1 using YAP1 antibodies

  • Immunoblot with ubiquitin antibodies to detect ubiquitination levels

  • Compare between ACAT2 knockdown and control cells

  • Research has shown that ACAT2 affects YAP1 ubiquitination via SETD7

• Proteasome Inhibition Studies:

  • Treat cells with proteasome inhibitors (e.g., MG132)

  • Compare YAP1 protein levels in ACAT2 knockdown vs. control cells

  • Western blot analysis to detect accumulated ubiquitinated proteins

• Ubiquitination Site Mapping:

  • Mass spectrometry analysis of immunoprecipitated YAP1

  • Site-directed mutagenesis of potential ubiquitination sites

  • Evaluate effect of ACAT2 on wild-type vs. mutant YAP1

• E3 Ligase Identification:

  • Screen candidate E3 ligases using siRNA libraries

  • Validate with co-immunoprecipitation between YAP1 and candidate E3 ligases

  • Assess how ACAT2 affects these interactions

What are common pitfalls in ACAT2 antibody experiments and how can they be addressed?

How should ACAT2 expression be interpreted in relationship to clinical outcomes?

Evidence-based interpretation requires:

How can ACAT2 antibodies be used in studying metabolic reprogramming in cancer?

ACAT2's role in metabolism can be investigated through:

• Metabolic Pathway Analysis:

  • Immunoprecipitate ACAT2 to identify interacting metabolic enzymes

  • Measure acetyl-CoA levels in ACAT2 knockdown vs. control cells

  • Assess the impact on lipid profiles using lipidomics approaches

• Epigenetic Regulation Mechanisms:

  • Investigate ACAT2's role in providing substrates (acetyl-CoA) for histone modifications

  • ACAT2 may contribute to epigenome programming by supplying metabolites used by enzymes involved in histone posttranslational modifications

  • Analyze histone acetylation patterns in ACAT2-manipulated cells

• Metabolic Inhibitor Combinations:

  • Test combinations of ACAT2 inhibitors with other metabolic pathway inhibitors

  • Evaluate synergistic effects on cancer cell viability and drug resistance

  • Monitor metabolic changes using NMR or mass spectrometry

What innovative approaches can be used to study ACAT2 in the tumor microenvironment?

Advanced methodological approaches include:

• Spatial Transcriptomics and Proteomics:

  • Combine ACAT2 antibody staining with spatial transcriptomics

  • Map ACAT2 expression patterns within the tumor microenvironment

  • Correlate with stromal and immune cell markers

• 3D Organoid Models:

  • Establish patient-derived organoids maintaining ACAT2 expression patterns

  • Immunostain for ACAT2 in different organoid regions

  • Test drug responses in organoids with varying ACAT2 levels

• Single-Cell Analysis:

  • Apply single-cell Western blotting for ACAT2

  • Combine with single-cell RNA sequencing to correlate protein and mRNA levels

  • Identify cell subpopulations with distinct ACAT2 expression patterns