ACOT6 Antibody

What is ACOT6 and what is its primary biological function?

ACOT6 (Acyl-CoA thioesterase 6) is an enzyme that functions as part of the acyl-CoA thioesterase family, which acts as a rheostat for intracellular levels of free fatty acids (FFAs) and fatty acyl-CoAs. These enzymes play a critical role in lipid metabolism by hydrolyzing acyl-CoAs to free fatty acids and coenzyme A. The human ACOT6 protein is encoded by the ACOT6 gene (Gene ID: 641372) and has a calculated molecular weight of approximately 23 kDa (207 amino acids) . ACOT6 is part of a larger family of ACOTs which are distributed in different subcellular compartments and have substrate specificity for various fatty acyl-CoAs, collectively forming an important regulatory system for cellular lipid homeostasis .

What types of ACOT6 antibodies are available for research, and what are their specific applications?

Several types of ACOT6 antibodies are available for research purposes. The polyclonal antibody (such as 22936-1-AP) can be used in multiple experimental applications including Western Blot (WB), Immunohistochemistry (IHC), and ELISA . This particular antibody shows reactivity with human, mouse, and rat samples, making it versatile for comparative studies across species. For Western blot applications, the recommended dilution range is 1:500-1:2000, while for immunohistochemistry applications, dilutions between 1:20-1:200 are recommended . Additionally, ELISA kits specific for human ACOT6 detection are available, utilizing sandwich ELISA methodology with a detection range of 0.313-20 ng/mL and a minimum detection limit of 0.313 ng/mL .

What tissues and cell types demonstrate significant ACOT6 expression?

According to validation data, ACOT6 expression has been confirmed in mouse liver tissue and HeLa cells through Western blot applications . Immunohistochemistry has also detected positive signals in human kidney tissue, suggesting expression in renal cells . Research investigating ACOT expression patterns during viral infection (specifically DENV2) has shown that ACOT6 expression can be modulated by the knockdown of other ACOT family members, indicating potential regulatory networks among these enzymes. For instance, knockdown of ACOT2 significantly reduced mRNA expression of ACOT6 in both mock-infected and DENV2-infected cells .

How can ACOT6 antibodies be utilized to study interactions between lipid metabolism and viral pathogenesis?

Recent research has revealed that ACOT enzymes, including ACOT6, play important roles in viral lifecycle regulation, particularly for flaviviruses like Dengue virus (DENV2). ACOT6 antibodies can be employed in immunoprecipitation and co-localization studies to investigate the physical interactions between viral components and lipid metabolism machinery. Experimental approaches would include using ACOT6 antibodies for protein detection in infected versus uninfected cells, combined with viral protein co-staining to determine spatial and temporal relationships .

Methodologically, researchers should prepare both mock-infected and virus-infected cell lysates, followed by immunoprecipitation using ACOT6 antibodies (using the recommended dilution of 1:500-1:2000 for Western blot detection) . This approach allows for the identification of virus-induced changes in ACOT6 protein-protein interactions. Additionally, immunofluorescence microscopy with ACOT6 antibodies can reveal subcellular redistribution of ACOT6 during infection, providing insights into how viruses manipulate lipid metabolic pathways .

What approaches should be considered when studying functional relationships between different ACOT family members using antibodies?

ACOT family members demonstrate complex functional relationships, with evidence suggesting compensatory or regulatory mechanisms among them. When studying these relationships, researchers should consider multiplex approaches combining ACOT6 antibodies with antibodies against other ACOT family members. Research has shown that knockdown of ACOT2 or ACOT7 affects the expression of other ACOTs, including ACOT6 .

For optimal results, design experiments with the following methodology: (1) Perform siRNA-mediated knockdown of specific ACOT family members, (2) Use ACOT6 antibodies in Western blot analysis (at 1:500-1:2000 dilution) to measure changes in protein levels , (3) Complement protein data with mRNA expression analysis using qRT-PCR, and (4) Compare results across different experimental conditions (e.g., with/without metabolic stress or viral infection). This integrated approach allows for comprehensive mapping of functional dependencies between ACOT6 and other family members .

How can ACOT6 antibodies contribute to understanding subcellular lipid metabolism compartmentalization?

ACOT enzymes are distributed across different subcellular compartments, including cytoplasm, mitochondria, and peroxisomes, creating a spatially organized system for lipid metabolism. ACOT6 antibodies can be instrumental in mapping this subcellular organization through immunofluorescence microscopy and subcellular fractionation studies.

The recommended methodological approach involves: (1) Performing subcellular fractionation to isolate different organelles, (2) Using Western blot with ACOT6 antibodies (1:500-1:2000 dilution) to analyze protein distribution across fractions , (3) Conducting co-localization studies combining ACOT6 antibodies with established organelle markers, and (4) Employing super-resolution microscopy for detailed spatial analysis. This multifaceted approach provides insights into how ACOT6 contributes to compartmentalized lipid metabolism and how this organization changes under different physiological or pathological conditions .

What are the optimal antigen retrieval methods for ACOT6 immunohistochemistry in different tissue types?

For effective ACOT6 detection in tissue samples using immunohistochemistry, proper antigen retrieval is critical. The recommended primary approach is antigen retrieval with TE buffer at pH 9.0, which has been validated for human kidney tissue samples . Alternatively, citrate buffer at pH 6.0 can also be used as an effective antigen retrieval method .

When optimizing your protocol, consider these methodological steps: (1) Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) retrieval methods with your specific tissue type, (2) Optimize incubation times (typically 15-30 minutes) and temperatures, (3) For each new tissue type, perform a dilution series of the antibody starting with the recommended range (1:20-1:200) to determine optimal signal-to-noise ratio , and (4) Include appropriate positive controls (such as human kidney tissue) and negative controls (primary antibody omission) in each experiment. Tissue-specific optimization is essential as antigen accessibility varies across different tissue types and fixation conditions .

What are the recommended sample preparation procedures for optimal ACOT6 detection in Western blot applications?

For effective ACOT6 detection using Western blot, sample preparation is a critical determinant of success. Based on validation data, the ACOT6 antibody (22936-1-AP) effectively detects the target protein at approximately 23 kDa in mouse liver tissue and HeLa cells .

The recommended sample preparation protocol includes: (1) Tissue or cell lysis using a buffer containing protease inhibitors to prevent protein degradation, (2) Sonication to ensure complete disruption of membranes and release of proteins, (3) Centrifugation to remove cellular debris, (4) Protein quantification to ensure equal loading, (5) Sample denaturation in loading buffer containing SDS and β-mercaptoethanol at 95-100°C for 5 minutes, and (6) Loading 20-40 μg of total protein per lane for cell lysates or 40-60 μg for tissue lysates. For electrophoresis, use 10-12% polyacrylamide gels to optimally resolve the 23 kDa ACOT6 protein, followed by transfer to PVDF or nitrocellulose membranes. For detection, use the antibody at a 1:500-1:2000 dilution, with the optimal dilution determined empirically for each sample type .

What controls should be included when validating ACOT6 knockdown or overexpression experiments?

When designing ACOT6 knockdown or overexpression experiments, proper controls are essential for result validation and interpretation. Based on research methodologies described for ACOT family members, the following controls should be included:

• For siRNA-mediated knockdown:

  • Non-target, irrelevant siRNA (IRR) as a negative control to account for off-target effects of siRNA treatment

  • Target-specific positive control siRNA (if available) to validate the knockdown methodology

  • Cytotoxicity assessment to ensure observed effects are not due to cell death

  • qRT-PCR verification of knockdown efficiency targeting ACOT6 mRNA levels

• For overexpression experiments:

  • Empty vector controls expressing the same selection marker

  • Western blot verification of overexpression using ACOT6 antibody (1:500-1:2000 dilution)

  • Functional assays to confirm the activity of the overexpressed protein

  • Time-course experiments to monitor expression stability

Additionally, when studying ACOT6 in relation to other ACOT family members, monitor the expression of related ACOTs (1, 2, 4, 7, 8, 9, 11, 12, 13) at both mRNA and protein levels to account for compensatory mechanisms or regulatory networks among these enzymes .

How should apparent molecular weight discrepancies for ACOT6 be interpreted in Western blot results?

These variations may result from several factors: (1) Post-translational modifications such as phosphorylation, glycosylation, or ubiquitination can increase the apparent molecular weight, (2) Alternative splicing may generate isoforms of different sizes, (3) Incomplete denaturation or disulfide bond formation can cause aberrant migration patterns, or (4) High salt concentration in samples can affect protein migration .

To troubleshoot molecular weight discrepancies: (1) Verify sample preparation procedures, ensuring complete denaturation, (2) Include verified positive controls such as mouse liver tissue or HeLa cell lysates, (3) Consider using gradient gels (4-20%) to better resolve proteins across a wider range of molecular weights, and (4) If consistent discrepancies are observed, consider validation with an alternative ACOT6 antibody or additional analytical techniques such as mass spectrometry .

What approaches can address non-specific binding when using ACOT6 antibodies in immunohistochemistry?

Non-specific binding can complicate the interpretation of immunohistochemistry results with ACOT6 antibodies. Based on the recommended protocols for ACOT6 antibody (22936-1-AP), several approaches can address this issue:

• Optimization of antibody dilution: Start with the recommended range (1:20-1:200) and test multiple dilutions to find the optimal concentration that maximizes specific signal while minimizing background .

• Blocking optimization: (a) Extend blocking time with 5-10% normal serum from the same species as the secondary antibody, (b) Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions, (c) Consider adding 0.1% BSA to the antibody diluent to reduce non-specific protein interactions.

• Antigen retrieval adjustment: Compare the recommended TE buffer (pH 9.0) with citrate buffer (pH 6.0) to determine which provides better signal-to-noise ratio for your specific tissue type .

• Additional controls: Include absorption controls (pre-incubating the antibody with excess antigen) to verify binding specificity, and isotype controls to identify potential non-specific binding of the antibody's constant region.

• Alternative detection systems: If peroxidase-based detection shows high background, consider fluorescence-based detection which may offer better signal discrimination .

How can conflicting data between ACOT6 protein levels and mRNA expression be reconciled?

Research with ACOT family members has sometimes revealed discrepancies between protein levels (detected by antibodies) and mRNA expression (measured by qRT-PCR). These discordances may reflect important biological regulatory mechanisms rather than experimental artifacts.

To reconcile such conflicting data: (1) Perform time-course experiments to identify potential temporal differences between mRNA expression and protein accumulation, (2) Investigate post-transcriptional regulation mechanisms such as miRNA-mediated repression or changes in mRNA stability, (3) Assess post-translational modifications and protein degradation pathways using proteasome or lysosome inhibitors, (4) Examine subcellular localization changes that might affect antibody accessibility while protein levels remain constant .

Methodologically, combine Western blot analysis using ACOT6 antibody (1:500-1:2000 dilution) with qRT-PCR, polysome profiling to assess translation efficiency, and pulse-chase experiments to measure protein stability . This integrated approach can reveal the mechanistic basis for apparent discrepancies between mRNA and protein abundance.

How does ACOT6 interact with other lipid metabolism enzymes in peroxisomal function?

Recent research suggests peroxisomal ACOTs (including ACOT4, ACOT6, and ACOT8) play crucial roles in the degradation of very long-chain fatty acids that cannot be directly shuttled to mitochondria . The expression of these peroxisomal ACOTs appears to be regulated by mitochondrial ACOTs such as ACOT2 and ACOT7, suggesting inter-organelle communication in lipid metabolism regulation .

Future research should investigate these interactions using co-immunoprecipitation with ACOT6 antibodies to identify protein-protein interactions, proximity ligation assays to visualize molecular proximities in situ, and functional assays measuring fatty acid metabolism in conditions where specific ACOTs are manipulated. Additionally, researchers should explore how these peroxisomal-mitochondrial interactions change under metabolic stress conditions or during viral infections, as peroxisomes are increasingly recognized as important mediators of viral infections and antiviral responses .

What role does ACOT6 play in viral lifecycle regulation and potential antiviral responses?

Research on ACOT family members has revealed complex roles in viral lifecycle regulation. While knockdown of ACOT1 enhanced dengue virus (DENV2) replication, knockdown of mitochondrial ACOTs (ACOT2 and ACOT7) suppressed viral replication . The specific role of ACOT6 in viral infection contexts is less characterized but appears to be influenced by other ACOT family members.

To investigate ACOT6's role in viral infections, researchers should: (1) Perform targeted ACOT6 knockdown experiments followed by viral infection, (2) Use ACOT6 antibodies (1:500-1:2000 dilution for Western blot) to monitor protein levels and localization changes during infection , (3) Analyze viral replication, protein translation, and particle infectivity following ACOT6 manipulation, and (4) Investigate the impact of ACOT6 on lipid droplet formation and composition during infection, as these are often critical for viral replication complex formation .

This research direction could reveal whether ACOT6 represents a potential target for antiviral therapeutic development, particularly for viruses that heavily depend on host lipid metabolism.

How can ACOT6 antibodies contribute to understanding cellular lipid homeostasis in disease states?

ACOT6 and other family members serve as a rheostat controlling intracellular levels of free fatty acids and fatty acyl-CoAs, suggesting potential roles in diseases characterized by dysregulated lipid metabolism . ACOT6 antibodies can be valuable tools for investigating these connections.

Promising research approaches include: (1) Comparative analysis of ACOT6 expression in normal versus diseased tissues using immunohistochemistry (1:20-1:200 dilution) , (2) Correlation of ACOT6 protein levels with disease progression or severity markers, (3) Investigation of ACOT6 post-translational modifications in disease states using phospho-specific or other modification-specific antibodies, and (4) Development of ACOT6-targeted interventions and monitoring their effects using antibody-based detection methods .