ACOT9 Antibody

What is ACOT9 and why is it important for metabolic research?

ACOT9 (Acyl-coenzyme A thioesterase 9) is a member of the acyl-CoA thioesterase superfamily, a group of enzymes that catalyze the hydrolysis of acyl-CoAs to free fatty acids and coenzyme A (CoASH). This mitochondrial protein plays a critical role in regulating intracellular levels of acyl-CoAs, free fatty acids, and CoASH. ACOT9 is particularly significant because it can simultaneously hydrolyze both short-chain and long-chain acyl-CoAs despite having markedly different Km values for these substrates . This dual functionality suggests ACOT9 provides a novel regulatory link between fatty acid and amino acid metabolism in mitochondria, making it an important target for metabolic research. Recent studies have also identified ACOT9 as having a strong association with obesity in humans, highlighting its potential importance in metabolism-related disorders .

What should researchers consider when selecting an ACOT9 antibody?

When selecting an ACOT9 antibody, researchers should carefully evaluate several parameters to ensure experimental success. First, verify species reactivity—commercially available antibodies like 15901-1-AP show reactivity with human, mouse, and rat samples . Second, confirm application compatibility based on your experimental methods; some antibodies are validated for Western blot (1:500-1:1000 dilution), immunohistochemistry (1:50-1:500), ELISA, and immunofluorescence applications . Third, consider the antibody type; most ACOT9 antibodies are rabbit polyclonal, which offers high sensitivity but may show batch-to-batch variation . Fourth, examine the immunogen information—for example, ABClonal's A15416 uses a recombinant fusion protein corresponding to amino acids 22-250 of human ACOT9 . Finally, check if the antibody detects the expected molecular weight (approximately 43-50 kDa for ACOT9) .

What is the subcellular localization of ACOT9 and how does this affect antibody-based detection methods?

ACOT9 primarily localizes to mitochondria but exhibits a dynamic distribution pattern between the inner mitochondrial membrane (IM) and matrix compartments depending on cellular conditions. Under basal conditions, ACOT9 is more abundant in the inner mitochondrial membrane, but it translocates into the matrix upon caloric stress and/or chronic cold exposure . This compartment shifting is functionally significant and appears to involve interaction with the electron transport chain Complex I subunit NDUFS7 . For antibody-based detection, this translocation phenomenon has important implications: immunofluorescence experiments should include mitochondrial markers to confirm colocalization, and subcellular fractionation protocols must carefully separate mitochondrial compartments to accurately track ACOT9 distribution. When performing immunostaining, researchers should consider that fixation methods may differentially preserve the native localization depending on the metabolic state of the cells or tissues being examined.

What are the validated applications for ACOT9 antibodies and their optimal protocols?

ACOT9 antibodies have been validated for multiple experimental applications, each requiring specific optimization. For Western blot (WB) applications, researchers have successfully used dilutions ranging from 1:500-1:2000, with recommended starting points of 1:500-1:1000 for Proteintech's 15901-1-AP antibody and 1:1000 for ABClonal's A15416 . Optimal protein loading is approximately 25μg per lane, with detection enhanced using HRP-conjugated secondary antibodies and ECL detection systems. For immunohistochemistry (IHC), dilutions of 1:50-1:500 are recommended, with antigen retrieval in TE buffer (pH 9.0) or alternatively citrate buffer (pH 6.0) . Immunofluorescence/immunocytochemistry (IF/ICC) applications work effectively at dilutions of 1:50-1:200, as demonstrated in A431 cells with Cy3-conjugated secondary antibodies . For all applications, appropriate blocking (e.g., 3% nonfat dry milk in TBST for WB) is essential to minimize background signal. The choice of fixative and permeabilization method should be optimized based on the specific cellular compartment being targeted, particularly considering ACOT9's dynamic mitochondrial localization.

How should researchers validate the specificity of ACOT9 antibodies in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For ACOT9 antibodies, a multi-tiered validation approach is recommended. First, conduct positive and negative control experiments using tissues or cell lines with known ACOT9 expression patterns; validated positive samples include HEK-293 cells, MCF-7 cells, mouse heart tissue, mouse kidney tissue, and rat kidney tissue . Second, verify the molecular weight of detected bands (expected at 43-50 kDa for ACOT9) . Third, consider using ACOT9 knockout or knockdown samples as negative controls; published studies using Acot9-/- mice provide excellent specificity controls . Fourth, when possible, test multiple antibodies targeting different epitopes of ACOT9—for example, compare antibodies raised against different regions of the protein such as the 15901-1-AP (targets a fusion protein) versus A15416 (targets amino acids 22-250) . Finally, consider orthogonal validation methods, such as confirming protein expression with mRNA levels via RT-PCR or demonstrating loss of signal after pre-incubation of the antibody with its immunizing peptide.

What tissue preparation and fixation protocols are optimal for ACOT9 detection in histological samples?

For optimal detection of ACOT9 in histological samples, tissue preparation and fixation protocols must preserve both antigenicity and mitochondrial morphology. For formalin-fixed paraffin-embedded (FFPE) sections, 10% neutral buffered formalin fixation for 24-48 hours, followed by standard paraffin embedding, is generally effective. Section thickness of 4-5μm is recommended for IHC applications. Importantly, ACOT9 detection requires optimization of antigen retrieval methods; the primary suggested method is heat-induced epitope retrieval (HIER) using TE buffer at pH 9.0, although citrate buffer at pH 6.0 can serve as an alternative . For frozen sections, fixation with 4% paraformaldehyde for 10-15 minutes followed by permeabilization with 0.2% Triton X-100 typically provides good results. When studying the submitochondrial localization of ACOT9 in research investigating its translocation between inner membrane and matrix compartments, electron microscopy with immunogold labeling may be necessary, requiring specialized fixation with glutaraldehyde/paraformaldehyde mixtures followed by careful dehydration and embedding in resin.

How does ACOT9 substrate specificity influence experimental design for activity assays?

ACOT9 exhibits a complex substrate specificity profile that requires careful consideration when designing activity assays. Research has demonstrated that ACOT9 can hydrolyze both short-chain and long-chain acyl-CoAs simultaneously despite having significantly different Km values for these substrates . When designing ACOT9 activity assays, researchers should consider including multiple substrate types, including short-chain acyl-CoAs (C2-C6), medium-chain acyl-CoAs (C8-C12), long-chain acyl-CoAs (C14-C20), and branched-chain CoA esters derived from amino acid metabolism. For acyl-CoA thioesterase activity assays, HPLC-based detection methods using UV detection at 260nm have been successfully employed, using an appropriate internal standard such as 3-hydroxy-3-methylglutaryl-CoA (which is not a substrate for ACOT9) . The reaction should be conducted in a suitable buffer system, with reactions terminated by acidification to pH 4 to inhibit ACOT9 activity. Additionally, researchers should be aware that ACOT9 activity is strongly regulated by NADH and CoA, so the concentration of these metabolites in the reaction mixture will significantly impact measured activity levels .

What approaches can be used to study ACOT9 submitochondrial translocation in response to metabolic stress?

Investigating ACOT9 submitochondrial translocation requires specialized approaches to distinguish between inner membrane and matrix localization. Based on recent findings that ACOT9 translocates from the inner membrane to the matrix upon caloric stress and chronic cold exposure , researchers can employ several complementary methods. First, subcellular fractionation with differential centrifugation can be used to isolate mitochondria, followed by further separation of mitochondrial compartments using digitonin treatment or osmotic shock techniques to separate outer membrane, inner membrane, and matrix fractions. Western blot analysis can then quantify ACOT9 distribution across these fractions, using compartment-specific markers (e.g., TOM20 for outer membrane, TIM23 for inner membrane, and matrix enzymes like citrate synthase) as controls. Second, super-resolution microscopy with co-localization studies using compartment-specific markers can visualize ACOT9 translocation in intact cells. Third, proximity labeling approaches like BioID or APEX2 fused to compartment-specific anchors can track ACOT9 movement in living cells. Finally, researchers should consider monitoring the association between ACOT9 and its binding partner NDUFS7 (a Complex I subunit), as this interaction may regulate the translocation process .

How can ACOT9's role in linking fatty acid and amino acid metabolism be investigated experimentally?

To investigate ACOT9's proposed role as a link between fatty acid and amino acid metabolism, researchers can implement multi-faceted experimental approaches. First, enzyme kinetic studies using recombinant ACOT9 and various substrates including fatty acyl-CoAs and CoA esters derived from amino acid catabolism can establish substrate preferences and potential competitive relationships. HPLC-based assays have been successfully used for this purpose, measuring the hydrolysis of substrates like C12-CoA and isobutyryl-CoA using 3-hydroxy-3-methylglutaryl-CoA as an internal standard . Second, metabolomic profiling of cells or tissues with ACOT9 overexpression or knockout (Acot9-/-) can reveal changes in metabolites from both pathways. Studies using Acot9-/- mice have already demonstrated effects on energy expenditure and adiposity . Third, stable isotope tracing experiments using labeled fatty acids or amino acids can track metabolic flux alterations when ACOT9 levels are manipulated. Fourth, protein interaction studies can identify ACOT9 binding partners involved in either pathway; NDUFS7 has already been identified as an interaction partner . Finally, tissue-specific knockout models, such as the BAT-specific knockout (Acot9B-KO) that affects body weight and fat mass, can help delineate tissue-specific functions .

What are common issues in ACOT9 antibody-based experiments and how can they be resolved?

When working with ACOT9 antibodies, researchers may encounter several technical challenges. One common issue is inconsistent detection of the expected 43-50 kDa band in Western blot applications. This may result from sample preparation conditions that disrupt mitochondrial integrity or protein degradation. To address this, use fresh samples, include protease inhibitors in all buffers, maintain samples at 4°C throughout processing, and optimize lysis conditions (e.g., RIPA buffer with 0.1% SDS for complete extraction). Another challenge is high background in immunostaining applications, which can be mitigated by optimizing blocking conditions (3% BSA or 5-10% normal serum from the same species as the secondary antibody), extending blocking time to 1-2 hours, and using more stringent washing protocols (e.g., 5x5 minute washes with 0.1% Tween-20 in PBS). For tissue-specific variations in signal intensity, researchers should adjust antibody concentrations based on the tissue type; kidney tissue typically shows strong ACOT9 expression and can serve as a positive control . If multiple bands appear in Western blots, this may reflect ACOT9 isoforms or post-translational modifications; confirmation with mass spectrometry or isoform-specific primers can help clarify the identity of these bands.

How can researchers integrate ACOT9 antibody data with functional studies of mitochondrial metabolism?

Integrating ACOT9 antibody-based detection with functional studies requires a coordinated experimental approach. Researchers can correlate ACOT9 protein levels or subcellular localization with functional readouts of mitochondrial metabolism using the following strategies: First, perform parallel analyses of ACOT9 expression (via Western blot) and mitochondrial respiration (using Seahorse XF analyzers or Clark-type oxygen electrodes) in the same samples under various conditions, such as nutrient deprivation or cold exposure known to trigger ACOT9 translocation . Second, combine immunofluorescence imaging of ACOT9 with live-cell mitochondrial functional probes (e.g., TMRM for membrane potential, MitoTracker for mitochondrial mass, or genetically-encoded sensors for ATP, ROS, or Ca2+) to correlate localization with functional parameters at the single-cell level. Third, use ACOT9 antibodies for immunoprecipitation followed by activity assays to link protein interaction partners with functional outcomes. Fourth, in animal models, correlate tissue-specific ACOT9 expression patterns with physiological parameters; for example, in Acot9B-KO mice, reduced BAT ACOT9 expression correlates with 13% lower body weight and 43% reduction in fat mass after HFD feeding compared to controls .

What considerations should be made when studying ACOT9 in disease models or patient samples?

When studying ACOT9 in disease models or patient samples, several important considerations should guide experimental design and interpretation. First, ACOT9 expression and localization may be altered in disease states, particularly those involving metabolic dysfunction; hence, careful selection of control samples and standardization of collection and processing protocols is essential. Second, given ACOT9's strong association with obesity in humans , researchers should comprehensively document metabolic parameters of study subjects or animal models, including body mass index, fat distribution, insulin sensitivity, and feeding status. Third, when analyzing patient samples, consider potential effects of medications, particularly those affecting mitochondrial function or metabolism (e.g., metformin, thiazolidinediones, statins) on ACOT9 expression or function. Fourth, for tissue biopsies, the timing of sample collection relative to fasting/feeding cycles is critical, as ACOT9 translocation is responsive to caloric stress . Finally, researchers should consider genetic variation in ACOT9 when studying diverse human populations; genetic analysis or sequencing may be necessary to identify functional variants that could affect antibody epitopes or protein function. For mouse models of human disease, note that while mouse and human ACOT9 share high homology, species-specific differences in regulation or interacting partners may exist.

How can advanced imaging techniques enhance ACOT9 localization and functional studies?

Advanced imaging technologies offer powerful approaches for investigating ACOT9's dynamic subcellular localization and function. Super-resolution microscopy techniques such as structured illumination microscopy (SIM), stimulated emission depletion (STED), or single-molecule localization methods (PALM/STORM) can resolve ACOT9 distribution within mitochondrial subcompartments at resolutions below 100 nm. These techniques can visualize the translocation of ACOT9 from the inner membrane to the matrix under stress conditions with unprecedented clarity . Live-cell imaging combined with ACOT9 fusion proteins (e.g., ACOT9-GFP) enables real-time tracking of translocation events in response to metabolic perturbations, though researchers must verify that the tag doesn't interfere with localization or function. Correlative light and electron microscopy (CLEM) can bridge the resolution gap between fluorescence microscopy and ultrastructural analysis, providing context for ACOT9 localization within the complex mitochondrial architecture. Förster resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) approaches can investigate ACOT9's interaction with identified binding partners like NDUFS7 in living cells. Finally, emerging proximity labeling techniques such as APEX2 or TurboID fused to ACOT9 can identify novel interaction partners in different submitochondrial compartments under various physiological conditions.

What genetic approaches complement antibody-based studies of ACOT9 function?

Genetic approaches provide powerful complementary strategies to antibody-based ACOT9 studies. CRISPR/Cas9 genome editing can generate precise knockout models in cell lines and animals, as demonstrated in the generation of Acot9-/- and tissue-specific knockout (e.g., Acot9B-KO) mouse models that revealed ACOT9's role in energy expenditure and adiposity regulation . For temporal control of ACOT9 expression, inducible knockdown or knockout systems (e.g., Tet-On/Off or tamoxifen-inducible Cre-loxP) allow researchers to distinguish between developmental and acute effects of ACOT9 loss. Point mutations targeting specific functional domains can dissect the structure-function relationships of ACOT9, particularly regarding its dual substrate specificity for short- and long-chain acyl-CoAs . Rescue experiments reintroducing wild-type or mutant ACOT9 into knockout backgrounds can confirm phenotype specificity and map functional domains. RNA-sequencing of tissues from Acot9-deficient models can reveal transcriptional networks affected by ACOT9 loss, while ribosome profiling might uncover translational effects. Notably, these genetic approaches should be designed with consideration of ACOT9's multiple isoforms and potential compensation by other acyl-CoA thioesterase family members.

How might metabolomic approaches extend our understanding of ACOT9 function beyond antibody detection?

Metabolomic approaches offer unique insights into ACOT9 function by directly measuring its impact on cellular metabolism. Targeted metabolomics focusing on acyl-CoA species, free fatty acids, and amino acid metabolites can quantify ACOT9's substrate and product pools in different tissues and under various physiological conditions. This is particularly relevant given ACOT9's ability to hydrolyze both fatty acyl-CoAs and CoA esters derived from amino acid metabolism, potentially providing a metabolic link between these pathways . Stable isotope tracing experiments using 13C-labeled fatty acids or amino acids can track metabolic flux alterations when ACOT9 levels are manipulated, revealing how ACOT9 activity redirects carbon flow through different pathways. Flux balance analysis integrating metabolomic data with computational modeling can predict system-wide effects of ACOT9 perturbation. Spatial metabolomics techniques such as MALDI imaging mass spectrometry can map metabolite distribution in tissues from wild-type versus Acot9-/- or Acot9B-KO mice, potentially correlating with ACOT9 expression patterns identified by immunohistochemistry . Finally, integration of metabolomic data with transcriptomic and proteomic datasets from the same experimental systems can provide a comprehensive multi-omics view of how ACOT9 influences cellular metabolism beyond what can be determined from antibody-based detection alone.