ACOT7 Antibody

What is ACOT7 and why is it important in research?

ACOT7 (acyl-CoA thioesterase 7) belongs to a group of enzymes that catalyze the hydrolysis of acyl-CoAs to free fatty acids and coenzyme A (CoASH), thus regulating intracellular levels of these metabolically important molecules . This protein preferentially hydrolyzes palmitoyl-CoA but demonstrates broad specificity for fatty acyl-CoAs with chain lengths ranging from C8 to C18 . ACOT7 appears to play a particularly important physiological role in brain function, as it is highly expressed in neural tissues . Recent research has identified ACOT7 as a potential biomarker for Alzheimer's disease, making antibodies against this protein valuable tools for neurodegenerative disease research . The protein exists in multiple isoforms with molecular weights between 27-40 kDa, with the predominant observed form appearing at approximately 37 kDa in Western blots .

ACOT7's importance extends beyond neurobiology, as it participates in fatty acid metabolism pathways that impact energy homeostasis and cellular signaling . The enzyme's ability to modulate lipid metabolism positions it as a critical research target for metabolic disorders, inflammatory conditions, and neurological diseases . Understanding ACOT7's tissue-specific distribution and subcellular localization provides insights into its diverse physiological functions and potential pathological implications.

Which applications are best supported by available ACOT7 antibodies?

Currently available ACOT7 antibodies have been validated for several common laboratory applications with varying levels of optimization . Western blotting (WB) is the most robustly supported application, with recommended dilutions ranging from 1:3000 to 1:50000 depending on the specific antibody formulation and sample type . Both polyclonal and monoclonal ACOT7 antibodies have demonstrated reliable performance in WB applications across multiple species including human, mouse, rat, rabbit, and pig samples .

Immunohistochemistry (IHC) and immunofluorescence (IF) applications are also supported by certain ACOT7 antibodies, though published validation data is less extensive than for WB . ELISA applications have been successfully implemented, particularly in recent Alzheimer's disease biomarker research where ACOT7 levels in serum were quantified . When selecting an antibody for a specific application, researchers should review the validation data for each specific antibody clone and consider factors such as species reactivity, epitope recognition, and documented performance in the intended application .

How should researchers optimize Western blot protocols for ACOT7 detection?

When optimizing Western blot protocols for ACOT7 detection, researchers should consider several key factors to ensure reliable and specific results. Begin with appropriate sample preparation, noting that ACOT7 is predominantly expressed in brain tissue, with liver mitochondria also showing clear expression of the 37 kDa band . For brain tissue samples, particular care should be taken during homogenization to preserve protein integrity .

For primary antibody incubation, start with the manufacturer's recommended dilution range (typically 1:3000-1:10000 for polyclonal and 1:5000-1:50000 for monoclonal antibodies) and optimize based on signal strength and background levels . Both polyclonal (e.g., 15972-1-AP) and monoclonal (e.g., 68140-1-Ig) antibodies have been validated for ACOT7 detection, with each offering different advantages in terms of specificity and sensitivity . When interpreting results, expect the predominant ACOT7 band at approximately 37 kDa, though faint bands at ~42 kDa and ~50 kDa may occasionally appear . It is worth noting that in certain tissues like brown adipose tissue (BAT), the antibody may generate multiple bands around 37 kDa, potentially complicating interpretation .

How can researchers distinguish between ACOT7 isoforms in experimental samples?

Distinguishing between ACOT7 isoforms presents a significant challenge due to their structural similarities and overlapping molecular weights. ACOT7 has at least seven identified isoforms ranging from 27-40 kDa, with three primary transcripts annotated in NCBI . Two of these transcripts (1 and 3) contain strongly predicted mitochondrial targeting sequences (MTS), while transcript 2 has a much lower prediction for mitochondrial localization . The unprocessed protein has a molecular weight of approximately 42 kDa, while cleavage of the N-terminal sequence in transcripts 1 and 3 yields a processed protein of approximately 37 kDa .

For researchers aiming to differentiate between these isoforms, several approaches can be implemented. First, subcellular fractionation techniques can help separate mitochondrial and cytosolic isoforms, as demonstrated in studies utilizing high-speed centrifugation to isolate mitochondrial fractions . Second, protease protection assays can provide insights into the submitochondrial localization of ACOT7, with experiments showing that the 37 kDa band is destabilized only when samples are treated with both protease and Triton X-100, suggesting matrix localization similar to pyruvate dehydrogenase (PDH) . Finally, researchers may employ isoform-specific primers for qPCR analysis or utilize mass spectrometry for definitive isoform identification when antibody-based methods prove insufficient.

What controls are essential when using ACOT7 antibodies in knockdown or knockout studies?

When conducting knockdown or knockout studies involving ACOT7, implementing appropriate controls is crucial for experimental validity and data interpretation. First and foremost, researchers should include both positive and negative controls in their antibody-based assays . Positive controls might include samples known to express ACOT7 at high levels, such as brain tissue or cell lines like Jurkat, HEK-293, or SK-N-SH cells . For negative controls, samples from ACOT7 knockout mice have been instrumental in validating antibody specificity, as demonstrated in previous studies .

For siRNA-mediated knockdown experiments, essential controls include non-targeting siRNA (siControl) to account for non-specific effects of the transfection procedure . Researchers should quantify knockdown efficiency through both protein (Western blot) and mRNA (qPCR) analyses, with effective ACOT7 knockdown typically achieving at least 80% reduction in expression levels . When studying the functional consequences of ACOT7 manipulation, it is advisable to measure multiple downstream targets or pathways, as exemplified by studies examining how ACOT7 silencing affects β-secretase (BACE1), Aβ42, amyloid precursor protein (APP), and βCTF levels . Additionally, rescue experiments, where ACOT7 expression is restored in knockout models, can provide compelling evidence for the specificity of observed phenotypes.

How does ACOT7 subcellular localization impact experimental design and interpretation?

ACOT7's subcellular localization has significant implications for experimental design and data interpretation. Research has demonstrated that ACOT7 can localize to both cytosolic and mitochondrial compartments, with tissue-specific distribution patterns . This dual localization necessitates careful consideration of sample preparation methods, particularly when studying tissue-specific functions or comparing ACOT7 activity across different tissues or experimental conditions.

When designing subcellular fractionation experiments, researchers should implement rigorous quality control measures to ensure clean separation of cellular compartments . Western blot analysis should include markers for relevant compartments (e.g., cytosolic, mitochondrial matrix, outer mitochondrial membrane) to verify fractionation efficiency . For mitochondrial studies, protease protection assays can help determine whether ACOT7 resides in the mitochondrial matrix, intermembrane space, or membrane compartments . The observation that liver mitochondria clearly express the 37 kDa ACOT7 band while other tissues show weaker mitochondrial expression highlights the importance of tissue-specific considerations in experimental design .

Immunofluorescence microscopy offers complementary insights into ACOT7 localization, though researchers should be aware that fixation and permeabilization methods can differentially affect the detection of cytosolic versus organelle-bound proteins . Colocalization studies with known compartment markers (e.g., MitoTracker for mitochondria) can provide additional evidence for ACOT7's distribution pattern, informing hypotheses about its tissue-specific functions.

How can ACOT7 antibodies be optimized for biomarker studies in Alzheimer's disease research?

Recent research has identified ACOT7 as a promising serum biomarker for Alzheimer's disease (AD), necessitating optimized antibody-based detection methods for clinical applications . When designing ELISA protocols for ACOT7 detection in serum samples, researchers should carefully consider several methodological aspects. First, antibody selection is crucial, with monoclonal antibodies often preferred for clinical biomarker applications due to their batch-to-batch consistency and specificity . The sensitivity of the assay should be sufficient to detect ACOT7 in the pg/mL range, as studies have established an optimal cut-off point of 62.5 pg/mL for distinguishing between AD patients and healthy controls .

Sample processing and storage conditions require standardization to ensure reproducible results across different laboratories and clinical settings . Serum samples should be collected following established protocols, and consistent freeze-thaw cycles should be maintained to prevent protein degradation . For validation studies, researchers should include both Western blot and ELISA analyses, as implemented in the study demonstrating elevated ACOT7 serum levels in AD patients (47% increase detected by Western blot, Control: 57.7 ± 20.6 pg/mL vs. AD: 99.0 ± 39.1 pg/mL by ELISA) . When evaluating ACOT7 as a diagnostic biomarker, receiver operating characteristic (ROC) curve analysis with calculation of area under the curve (AUC), sensitivity, specificity, and diagnostic accuracy provides essential metrics for assessing clinical utility .

What methodological approaches can address cross-reactivity issues with ACOT7 antibodies?

Cross-reactivity remains a significant concern when working with antibodies against members of protein families with high sequence homology, such as acyl-CoA thioesterases. To address this challenge with ACOT7 antibodies, researchers should implement several methodological approaches. First, antibody validation using tissues or cells from ACOT7 knockout models provides the gold standard for specificity confirmation . Published studies have utilized custom-made antibodies specifically tested in tissues from Acot7 knockout mice to ensure specificity .

When knockout samples are unavailable, peptide competition assays can help assess antibody specificity. In these experiments, pre-incubation of the antibody with the immunizing peptide should abolish specific signals while leaving non-specific signals intact . Additionally, comparing results from multiple antibodies recognizing different epitopes of ACOT7 can increase confidence in the specificity of observed signals . For tissues known to express multiple ACOT7 isoforms or where antibody performance is suboptimal (e.g., BAT, where the antibody generates multiple bands around 37 kDa), alternative detection methods such as mass spectrometry or targeted PCR should be considered .

Researchers should also be aware that some antibodies perform better in specific applications or with particular species . For instance, the polyclonal antibody 15972-1-AP has demonstrated reactivity with human, mouse, and rat samples, while the monoclonal antibody 68140-1-Ig shows broader species reactivity including human, rat, mouse, rabbit, and pig samples . Thorough review of validation data and published literature can guide selection of the most appropriate antibody for specific experimental contexts.

How can ACOT7 knockdown studies inform our understanding of Alzheimer's disease pathophysiology?

ACOT7 knockdown studies have provided valuable insights into this protein's potential role in Alzheimer's disease (AD) pathophysiology, particularly regarding its influence on amyloid precursor protein (APP) metabolism . When designing knockdown experiments to investigate ACOT7's role in AD, researchers should consider using neuronal cell lines that express APP, such as SK-N-SH APPwt cells . Effective siRNA-mediated knockdown of ACOT7 (achieving approximately 88% reduction in expression) has been shown to significantly impact key components of the amyloidogenic pathway .

Methodologically, researchers should assess multiple parameters following ACOT7 knockdown, including levels of β-secretase (BACE1), Aβ42, APP, and βCTF . The observed decreases in these components after ACOT7 silencing suggest that ACOT7 modulates the amyloidogenic pathway of APP metabolism without affecting the non-amyloidogenic pathway . This finding aligns with clinical observations of elevated ACOT7 levels in AD patients and the negative correlation between serum ACOT7 and Mini-Mental State Examination (MMSE) scores (r = -0.85, p < 0.001) .

For comprehensive analysis, researchers should combine protein-level assessments with functional assays, such as measuring secreted Aβ42 in cell culture media or evaluating downstream signaling pathways affected by altered APP processing . Time-course experiments can provide additional insights into whether ACOT7's effects are immediate or require longer-term metabolic adaptations. These methodological approaches can help elucidate the mechanistic link between ACOT7 and AD pathophysiology, potentially identifying new therapeutic targets or diagnostic strategies.

What strategies can resolve multiple banding patterns when using ACOT7 antibodies?

Multiple banding patterns are a common challenge when working with ACOT7 antibodies, particularly in certain tissues or experimental conditions . To address this issue, researchers can implement several technical strategies. First, optimize sample preparation to minimize protein degradation or modification, as these can contribute to unexpected banding patterns . Fresh sample preparation, inclusion of appropriate protease inhibitors, and consistent handling procedures can help reduce artifactual bands .

Adjusting electrophoresis conditions can improve band resolution and separation. Using gradient gels (e.g., 4-12% or 4-20%) may help distinguish between closely migrating ACOT7 isoforms with similar molecular weights . For tissues known to produce complex banding patterns, such as brown adipose tissue (BAT), which was excluded from ACOT7 analysis in some studies due to multiple bands around 37 kDa, alternative approaches may be necessary . These might include using different antibody clones, optimizing blocking conditions to reduce non-specific binding, or implementing more stringent washing protocols .

When multiple bands persist despite optimization efforts, additional validation experiments become essential. These might include mass spectrometry analysis of the bands in question, parallel analysis with multiple ACOT7 antibodies targeting different epitopes, or comparison with samples from ACOT7 knockout models . Researchers should also consider the possibility that some bands represent legitimate ACOT7 isoforms or post-translationally modified variants, as ACOT7 has seven documented isoforms between 27-40 kDa .

How should researchers address tissue-specific variations in ACOT7 expression and antibody performance?

ACOT7 expression varies significantly across tissues, with particularly high levels in brain and testis, necessitating tissue-specific optimization of antibody-based detection methods . When working with brain tissue, researchers should expect strong ACOT7 signals at approximately 37 kDa, reflecting the high expression in this tissue . For liver samples, mitochondrial fractions show clear expression of the 37 kDa band, while other tissues may exhibit weaker mitochondrial signals . Brown adipose tissue (BAT) presents particular challenges, with the ACOT7 antibody generating multiple bands around 37 kDa that complicate interpretation .

To address these tissue-specific variations, several approaches can be implemented. First, adjust antibody dilutions based on expected expression levels, using higher dilutions (e.g., 1:10000 or greater) for high-expressing tissues like brain to prevent signal saturation . For tissues with lower expression, more concentrated antibody solutions and enhanced detection systems may be necessary . Second, optimize loading amounts based on preliminary experiments to determine the range of ACOT7 expression across different tissues . Third, consider subcellular fractionation to enrich for compartments where ACOT7 is predominantly localized in specific tissues (e.g., cytosolic versus mitochondrial fractions) .

For normalization and quantification, select loading controls appropriate for the tissue and subcellular fraction being analyzed . Studies have employed multiple normalization proteins, such as the 37 kDa subunit of Complex I, SDHA, and cytochrome c, using the average of normalized values to enhance reliability . When comparing ACOT7 expression across tissues, parallel processing of samples under identical conditions is essential for meaningful comparisons.

What experimental considerations are important when using ACOT7 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) studies using ACOT7 antibodies require careful experimental design to identify authentic protein-protein interactions while minimizing artifacts. When planning Co-IP experiments with ACOT7, antibody selection is a critical first step. Both polyclonal and monoclonal antibodies have been used for ACOT7 detection, but their suitability for immunoprecipitation may vary . Polyclonal antibodies often provide higher affinity and can recognize multiple epitopes, potentially enhancing precipitation efficiency, while monoclonal antibodies offer higher specificity that can reduce background .

Lysis buffer composition significantly impacts Co-IP success by affecting protein solubilization and preservation of native interactions . For ACOT7, which interacts with lipid substrates and potentially membrane-associated proteins, detergent selection is particularly important . Non-ionic detergents like NP-40 or Triton X-100 at moderate concentrations (0.5-1%) often provide a good balance between protein extraction and interaction preservation . Pre-clearing lysates with appropriate control beads (e.g., Protein A/G without antibody) can reduce non-specific binding .

Validation controls are essential for interpreting Co-IP results. These should include "no antibody" controls, isotype-matched control antibodies, and, when possible, samples from ACOT7 knockout or knockdown models . For detection of co-precipitated proteins, consider potential cross-reactivity between secondary antibodies used for detection and the immunoprecipitating antibody . To minimize this issue, researchers can use antibody-free detection methods or specialized secondary antibodies that recognize only native (non-denatured) primary antibodies.

How might ACOT7 antibodies contribute to understanding brain lipid metabolism in neurological disorders?

ACOT7 antibodies hold significant potential for advancing our understanding of brain lipid metabolism in neurological disorders, given ACOT7's high expression in neural tissues and its recently established connection to Alzheimer's disease . Future research could employ ACOT7 antibodies to map the protein's expression patterns across different brain regions and cell types in both healthy and diseased states . Immunohistochemistry and immunofluorescence approaches using validated ACOT7 antibodies could reveal altered distribution patterns in conditions like AD, Parkinson's disease, or traumatic brain injury .

Beyond descriptive studies, functional investigations could utilize ACOT7 antibodies to identify protein interaction networks through co-immunoprecipitation followed by mass spectrometry . Such approaches might reveal previously unknown binding partners that connect ACOT7 to specific neurological disease pathways . Additionally, antibody-based enzyme activity assays could help determine whether ACOT7's catalytic function is altered in neurological disorders, potentially revealing new therapeutic targets .

The recent finding that ACOT7 levels are elevated in the serum of AD patients and correlate with disease severity (as measured by MMSE scores) suggests that ACOT7 antibodies may also be valuable for developing diagnostic or prognostic assays . Future research might explore whether ACOT7 levels change longitudinally during disease progression or in response to therapeutic interventions . The demonstrated role of ACOT7 in modulating the amyloidogenic pathway of APP metabolism further supports investigating how this enzyme contributes to the pathophysiology of neurodegenerative conditions characterized by protein aggregation .

What novel methodologies might enhance ACOT7 detection sensitivity and specificity in complex biological samples?

As research into ACOT7's biological functions and clinical relevance advances, developing enhanced detection methodologies becomes increasingly important. Several emerging approaches show promise for improving ACOT7 detection in complex biological samples. Proximity ligation assays (PLA) could significantly enhance sensitivity and specificity by requiring dual antibody recognition for signal generation . This approach would be particularly valuable for detecting low-abundance ACOT7 in clinical samples or for visualizing ACOT7 interactions with other proteins in situ .

Mass spectrometry-based targeted proteomics offers another advanced approach for ACOT7 quantification . Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) methods could provide absolute quantification of ACOT7 and its isoforms without relying on antibody recognition . This approach would be especially valuable for distinguishing between closely related ACOT7 isoforms that may be difficult to resolve using antibody-based methods alone .

For clinical applications, particularly in AD biomarker development, digital ELISA technologies like Single Molecule Array (Simoa) could substantially lower detection limits compared to conventional ELISA . Given that the optimal cut-off value for ACOT7 in AD diagnosis was determined to be 62.5 pg/mL, ultrasensitive detection methods could improve diagnostic accuracy, especially for early disease stages . Additionally, multiplexed immunoassays that simultaneously measure ACOT7 alongside established biomarkers (e.g., Aβ42/40 ratio) could enhance diagnostic performance through multi-parameter algorithms .

How can ACOT7 antibodies be utilized in investigating metabolic crosstalk between neurons and glia in neurodegenerative diseases?

The metabolic interplay between neurons and glial cells represents a frontier in neurodegenerative disease research, and ACOT7 antibodies offer valuable tools for investigating this complex relationship . ACOT7's role in fatty acid metabolism and its high expression in brain tissue position it as a potential mediator of metabolic crosstalk between neural cell types . Future research could employ dual immunofluorescence labeling with ACOT7 antibodies alongside cell type-specific markers to map expression patterns across neurons, astrocytes, microglia, and oligodendrocytes in health and disease states .

Laser capture microdissection combined with immunohistochemistry and subsequent proteomic or transcriptomic analysis could reveal cell type-specific alterations in ACOT7 expression or localization in neurodegenerative conditions . This approach would be particularly valuable for understanding whether the elevated serum ACOT7 levels observed in AD patients reflect changes in specific brain cell populations . Additionally, in vitro co-culture systems using neurons and glia could be combined with ACOT7 knockdown or overexpression to investigate how this enzyme influences intercellular lipid trafficking and metabolism .

The recent finding that ACOT7 modulates the amyloidogenic pathway of APP metabolism suggests it may influence neuronal-glial interactions in AD pathogenesis . Immunoprecipitation studies using ACOT7 antibodies could help identify whether the protein participates in complexes that regulate metabolite exchange between cell types . Furthermore, in vivo studies using cell type-specific ACOT7 knockout models, validated using appropriate antibodies, could provide insights into how this enzyme contributes to metabolic homeostasis in the brain and how its dysregulation might promote neurodegenerative processes .