ACOT2 Antibody

What is ACOT2 and what cellular functions does it serve?

ACOT2 (acyl-CoA thioesterase 2) is a mitochondrial enzyme that hydrolyzes long-chain acyl-CoAs to free fatty acids and coenzyme A, functioning as a critical regulator of fatty acid metabolism. The protein has a calculated molecular weight of 53 kDa (483 amino acids) though it is typically observed at 46-53 kDa in experimental contexts . ACOT2 serves as a metabolic rheostat that controls the intracellular levels of free fatty acids and fatty acyl-CoAs, particularly within mitochondria. Unlike its cytoplasmic homolog ACOT1 (which shares approximately 94% sequence homology), ACOT2 contains a unique N-terminal mitochondrial targeting sequence that differentiates its cellular localization and specific functions . Recent research indicates that ACOT2 plays unexpected roles in viral replication pathways, with knockdown studies demonstrating that ACOT2 functionality is critical for the dengue virus lifecycle, potentially through its effects on viral protein translation and particle infectivity .

What applications are ACOT2 antibodies suitable for?

ACOT2 antibodies have been validated for multiple experimental applications with specific dilution recommendations for each technique. Based on the available data, these antibodies can be reliably used in:

For optimal results, researchers should titrate the antibody concentration in each specific experimental system, as detection efficiency can vary based on target protein abundance and sample characteristics .

What are the optimal storage conditions for maintaining ACOT2 antibody activity?

ACOT2 antibodies are typically supplied in a liquid formulation containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For maximum stability and activity retention, these antibodies should be stored at -20°C, where they remain stable for approximately one year after shipment . Importantly, the high glycerol content (50%) makes aliquoting unnecessary for -20°C storage, which reduces the risk of contamination and freeze-thaw degradation . Some preparations may contain 0.1% BSA as a stabilizer, particularly in smaller (20 μl) size formats . Researchers should note that while ambient temperature shipping is generally acceptable, prolonged exposure to higher temperatures should be avoided to prevent antibody degradation .

How can researchers differentiate between ACOT1 and ACOT2 in experimental systems?

Differentiating between ACOT1 and ACOT2 presents a significant challenge due to their high sequence homology (approximately 94%) . The primary structural difference is the mitochondrial targeting sequence present in ACOT2 but absent in the cytosolic ACOT1. To specifically detect ACOT2:

• Subcellular fractionation approach: Isolate mitochondrial fractions before immunoblotting to enrich for ACOT2 while reducing ACOT1 signals.

• Selective antibodies: Use antibodies raised against epitopes in the N-terminal region (amino acids 1-30) which contains the mitochondrial targeting sequence unique to ACOT2.

• RNA interference validation: When utilizing siRNA knockdown approaches, design ACOT2-specific siRNAs targeting the unique mitochondrial targeting sequence region. Importantly, researchers should validate knockdown specificity through qRT-PCR with primers that can distinguish between the two transcripts .

• Immunofluorescence co-localization: Employ co-staining with mitochondrial markers (e.g., MitoTracker) to confirm mitochondrial localization of the detected signal, characteristic of ACOT2 rather than cytosolic ACOT1.

Research has demonstrated that ACOT1 and ACOT2 can have differential effects on cellular processes despite their similar substrate specificity. For example, in dengue virus replication studies, combined knockdown of ACOT1 and ACOT2 increased viral replication by approximately 470%, while ACOT2-specific knockdown reduced viral replication by about 50% , indicating distinct functional roles.

What methodological considerations are important for optimizing ACOT2 immunohistochemistry?

For successful ACOT2 immunohistochemistry, several critical methodological factors must be considered:

• Antigen retrieval protocol: ACOT2 detection in FFPE tissues requires specific antigen retrieval conditions. The recommended primary approach is TE buffer at pH 9.0, though citrate buffer at pH 6.0 can serve as an alternative if optimization is needed . This step is crucial for exposing the epitope that may be masked during fixation.

• Antibody dilution optimization: A tiered dilution series ranging from 1:50 to 1:500 should be tested on relevant positive control tissues, with human kidney, skeletal muscle, heart, and testis serving as reliable positive controls .

• Detection system selection: For mitochondrial proteins like ACOT2, high-sensitivity detection systems such as polymer-based detection or tyramide signal amplification may improve signal-to-noise ratios, particularly in tissues with high autofluorescence.

• Blocking protocol refinement: Due to ACOT2's involvement in fatty acid metabolism, tissues with high lipid content may require extended blocking steps (5-10% normal serum with 0.1-0.3% Triton X-100) to reduce nonspecific binding.

• Multi-tissue validation: ACOT2 antibody has been validated across diverse human tissues including kidney, skeletal muscle, heart, testis, liver, spleen, and ovary , making these appropriate positive controls for antibody validation prior to testing experimental samples.

For mitochondrial co-localization studies in IHC applications, sequential staining with mitochondrial markers followed by ACOT2 antibody may provide conclusive evidence of proper target detection.

How can researchers effectively validate ACOT2 knockdown in functional studies?

Validating ACOT2 knockdown requires a multi-faceted approach to ensure specificity and effectiveness:

• Transcript quantification via qRT-PCR: Design primers that specifically distinguish ACOT2 from ACOT1 (focusing on the mitochondrial targeting sequence region). Normalize ACOT2 expression to stable reference genes such as RPLP0, which has been validated in this context . Calculate knockdown efficiency using the delta-delta Cq method compared to irrelevant controls.

• Protein-level validation: Western blotting using ACOT2-specific antibodies at 1:500-1:1000 dilution can confirm reduced protein expression. Expected molecular weight is 46-53 kDa .

• Functional validation: Since ACOT2 regulates mitochondrial fatty acid metabolism, researchers can measure changes in:

  • Mitochondrial fatty acid oxidation rates

  • Acyl-CoA:free fatty acid ratios in mitochondrial fractions

  • Oxygen consumption rates specific to fatty acid substrates

• Subcellular localization control: Immunofluorescence staining of knockdown cells (1:50-1:500 dilution) co-stained with mitochondrial markers can visually confirm reduced ACOT2 signal specifically in mitochondria .

• Phenotypic validation: Based on known ACOT2 functions, knockdown cells should show altered responses to metabolic challenges. For example, in viral infection models, ACOT2 knockdown significantly reduces viral protein translation by approximately 50% and decreases infectious virus release by approximately 76% .

Importantly, researchers must include appropriate controls including irrelevant siRNA controls to account for off-target effects of siRNA treatment .

What role does ACOT2 play in viral replication, and how can antibodies help investigate this function?

Recent research has revealed an unexpected and critical role for ACOT2 in viral replication processes, particularly for dengue virus (DENV2). The relationship between ACOT2 and viral replication offers a novel research direction:

• ACOT2 knockdown effects: siRNA-mediated specific knockdown of ACOT2 in Huh7 cells results in:

  • Significant reduction (~76%) in infectious DENV2 virus release

  • Reduction of viral genome replication by approximately 50%

  • Reduction in viral protein translation by ~50%

  • Increase in the viral particle/plaque-forming unit ratio, indicating decreased particle infectivity

• Methodological approaches using antibodies:

  • Immunofluorescence microscopy (1:50-1:500 dilution) can track ACOT2 localization changes during viral infection

  • Western blotting (1:500-1:1000) can monitor ACOT2 expression levels throughout the viral lifecycle

  • Immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg of total protein) can identify virus-host protein interactions involving ACOT2

• Differential effects from ACOT1 vs ACOT2: Interestingly, combined knockdown of ACOT1 and ACOT2 increases viral replication (~470%) and infectious viral release (~178%), while ACOT2-specific knockdown has the opposite effect . This suggests that these homologous proteins play antagonistic roles in viral replication processes.

• Mechanistic investigations: The data suggest that ACOT2's mitochondrial function may impact viral assembly and maturation processes rather than just replication, as evidenced by the decreased particle infectivity following ACOT2 knockdown.

Researchers investigating viral-host interactions can use ACOT2 antibodies to explore how this mitochondrial thioesterase's regulation of fatty acid metabolism interfaces with viral replication machinery, potentially identifying new therapeutic targets.

What troubleshooting approaches are recommended for inconsistent ACOT2 antibody results?

When encountering inconsistent results with ACOT2 antibodies, researchers should systematically address potential issues:

• Epitope accessibility assessment: ACOT2's mitochondrial localization may limit epitope accessibility. If signal is weak:

  • For WB: Ensure complete membrane protein solubilization (consider stronger lysis buffers containing 1-2% SDS)

  • For IHC/IF: Test alternative antigen retrieval methods; the recommended TE buffer at pH 9.0 is preferred, though citrate buffer at pH 6.0 is an acceptable alternative

• Cross-reactivity mitigation:

  • Increase antibody dilution (test range from 1:500-1:1000 for WB, 1:50-1:500 for IHC/IF)

  • Include additional blocking steps with 5% BSA or 5% milk

  • For WB applications, run longer SDS-PAGE to better separate ACOT2 (46-53 kDa) from potential cross-reactive proteins

• Sample preparation optimization:

  • For mitochondrial proteins like ACOT2, ensure samples are freshly prepared to preserve structural integrity

  • Consider subcellular fractionation to enrich for mitochondrial content before analysis

  • For cell lines, test multiple positive controls known to express ACOT2 (HEK-293, HepG2, human brain tissue, human kidney tissue)

• Detection system refinement:

  • For IF/ICC applications, consider signal amplification methods when working with low-expressing samples

  • For flow cytometry, ensure adequate permeabilization (0.80 μg antibody per 10^6 cells)

  • For WB, optimize transfer conditions for proteins in the 46-53 kDa range

• Validation with alternative antibodies: Consider using antibodies targeting different epitopes of ACOT2 to confirm results - options include antibodies targeting the middle region, internal region, or specific amino acid sequences (aa 222-483 or aa 360-409) .

Each troubleshooting approach should be methodically documented to build a laboratory-specific protocol for consistent ACOT2 detection across experimental systems.