ABHD6 Antibody, HRP conjugated

What is ABHD6 and what role does it play in cellular physiology?

ABHD6 is a serine hydrolase belonging to the α/β-hydrolase fold superfamily that functions primarily as a monoacylglycerol lipase. It preferentially hydrolyzes medium-chain saturated monoacylglycerols, including the endocannabinoid 2-arachidonoylglycerol (2-AG). Through 2-AG degradation, ABHD6 regulates endocannabinoid signaling pathways that control neurotransmission and neuroinflammation. Research indicates that ABHD6 may also possess lysophosphatidyl lipase activity with preference for lysophosphatidylglycerol among other lysophospholipids . ABHD6 has been established as a rate-limiting step in 2-AG signaling, making it a bona fide member of the endocannabinoid signaling system with significant implications for neurological and metabolic processes .

What is the tissue distribution and expression pattern of ABHD6?

ABHD6 shows variable expression across different tissues and cell types. Quantitative PCR analysis has revealed that ABHD6 mRNA is abundantly expressed in adult mouse brain and neurons in primary culture, with lower expression in microglia. The table below summarizes the relative expression levels across different neural tissues:

This distribution pattern suggests that ABHD6 plays a more significant role in neuronal function compared to glial cells in the brain . Outside the central nervous system, ABHD6 is also expressed in peripheral tissues including liver, adipose tissue, and kidney, with particularly high expression in the liver as demonstrated in antisense oligonucleotide (ASO) knockdown studies .

What are the key specifications of ABHD6 antibody, HRP conjugated, and how should it be stored?

ABHD6 antibody, HRP conjugated, is designed for enhanced detection sensitivity in various applications, particularly ELISA. According to product specifications, the antibody has a molecular weight between 35-40 kDa and recognizes ABHD6 protein (calculated molecular weight: 38 kDa from 337 amino acids) . The antibody is typically supplied in liquid form with a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

For optimal stability, the antibody should be stored at -20°C, where it remains stable for one year after shipment. Notably, aliquoting is unnecessary for -20°C storage for small volume formats (20μl sizes often contain 0.1% BSA as a stabilizer) . When working with the antibody, it's important to avoid repeated freeze-thaw cycles, which can degrade antibody quality and affect performance in experimental applications. Proper storage conditions maintain antibody specificity and reactivity with human, mouse, and rat samples as indicated in product specifications .

What are the optimal dilutions and applications for ABHD6 antibody, HRP conjugated?

ABHD6 antibody, HRP conjugated, has been validated for Western blot (WB) and ELISA applications. For Western blotting, the recommended dilution range is 1:5000-1:50000, though optimal dilution should be determined for each specific experimental system . This wide dilution range indicates the antibody's high sensitivity, allowing researchers to conserve antibody while maintaining robust detection.

For ELISA applications, similar dilution ranges can be used as starting points, but optimization is essential for quantitative results. The antibody has demonstrated positive detection in various cell lines including HeLa, A549, THP-1, COLO 320, C6, and RAW 264.7 cells, making these suitable positive controls for validation . The HRP conjugation eliminates the need for secondary antibodies, streamlining protocols and potentially reducing background. When optimizing protocols, researchers should consider that sample-dependent factors may influence optimal antibody concentration, and validation in each specific experimental context is recommended.

What methodological approaches can verify ABHD6 antibody specificity?

Verifying antibody specificity is crucial for ensuring reliable research results. Several complementary approaches can be employed to confirm ABHD6 antibody specificity:

• Genetic validation: Comparing antibody detection between wildtype samples and those with ABHD6 knockdown or knockout provides robust evidence of specificity. Studies have demonstrated that ABHD6 knockdown using antisense oligonucleotides reduced ABHD6 mRNA by >80% after 4 weeks, providing a useful validation system . Conditional knockout models, such as NAc-specific ABHD6 deletion verified by immunostaining, also serve as excellent specificity controls .

• Multiple antibody comparison: Using different antibodies targeting distinct ABHD6 epitopes should yield consistent results if they are specific. Comparing monoclonal and polyclonal antibodies can provide complementary evidence of target recognition.

• Blocking peptide assays: Pre-incubating the antibody with the immunizing peptide should abolish specific binding, providing evidence of epitope specificity.

• Western blot molecular weight verification: ABHD6 antibodies should detect a protein with observed molecular weight between 35-40 kDa, consistent with the calculated 38 kDa molecular weight of ABHD6 .

These validation approaches should be employed systematically when introducing a new ABHD6 antibody into a research program, particularly when working with new tissue types or experimental conditions.

How does ABHD6 regulate 2-AG signaling and what experimental approaches can demonstrate this function?

ABHD6 regulates 2-AG signaling by controlling its accumulation and efficacy at cannabinoid receptors. As a postsynaptically localized enzyme, ABHD6 limits the availability of 2-AG before it can act on presynaptic cannabinoid receptors. Several experimental approaches can demonstrate this regulatory function:

• Enzyme inhibition studies: Using selective ABHD6 inhibitors like WWL70 has been shown to increase 2-AG levels and enhance cannabinoid receptor activation. Research has demonstrated that WWL70 dose-dependently inhibits [³H]-2-AG hydrolysis in ABHD6-expressing systems without affecting MAGL activity, confirming ABHD6's direct role in 2-AG metabolism .

• Gene silencing experiments: Knockdown of ABHD6 using shRNA or other genetic approaches enhances 2-AG-induced effects. For instance, BV-2 cells with ABHD6 knocked down showed approximately 3-fold increased migration in response to 2-AG compared to only 2-fold in control cells or cells with FAAH knocked down . This enhanced response could be blocked by cannabinoid receptor antagonists like SR144528, confirming the mechanism involves increased 2-AG action at these receptors.

• Metabolomic profiling: Measuring changes in 2-AG levels and related lipids following ABHD6 manipulation provides direct evidence of its role in endocannabinoid metabolism.

These approaches collectively establish ABHD6 as a crucial regulator of 2-AG signaling by controlling its availability at cannabinoid receptors despite accounting for a relatively small percentage of total 2-AG hydrolysis in the brain.

How do conditional ABHD6 knockout models contribute to understanding tissue-specific functions?

Conditional knockout models have been instrumental in elucidating ABHD6's tissue-specific functions by allowing selective deletion in specific cell types or brain regions. These models provide several advantages over global knockout approaches:

• Spatial specificity: Conditional deletion in specific regions, such as the nucleus accumbens (NAc), allows researchers to attribute observed phenotypes to ABHD6 function in that particular region. For example, viral-mediated conditional deletion of ABHD6 in NAc neurons has been achieved with specificity verified by immunostaining .

• Cell-type specificity: Neuronal-specific conditional deletion approaches allow researchers to distinguish between ABHD6's role in neurons versus glia. This is critical because complete loss of ABHD6 in all cell types might mask cell type-specific effects or lead to compensatory mechanisms.

• Pathway analysis: Conditional NAc neuronal ABHD6 loss-of-function has been associated with reduced expression of cannabinoid receptor 1 (Cnr1) without impacting monoacylglycerol lipase (Mgll) expression . This suggests region-specific effects on the broader endocannabinoid system that might not be evident in global knockout models.

• Developmental considerations: Conditional knockouts can be induced at specific developmental timepoints, allowing researchers to distinguish between developmental roles of ABHD6 and its functions in adult tissues.

When designing studies with conditional ABHD6 knockout models, researchers should carefully consider the efficiency and specificity of the deletion approach, as studies have noted that neuron-specific conditional deletion approaches do not typically result in complete loss of the target protein in the specified region .

How does ABHD6 inhibition impact related signaling pathways beyond 2-AG metabolism?

ABHD6 inhibition has broader effects on signaling pathways beyond its direct impact on 2-AG metabolism, revealing complex interconnections within the endocannabinoid system and beyond:

• Effects on anandamide metabolism: Conditional ABHD6 knockout in NAc neurons resulted in reduced expression of both fatty acid amide hydrolase (FAAH) and N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD), which are responsible for degrading and synthesizing anandamide, respectively . This suggests cross-talk between 2-AG and anandamide signaling pathways.

• Metabolic pathway modulation: ABHD6 knockdown in liver significantly reduced high-fat diet-induced hepatic triacylglycerol accumulation and suppressed expression of key lipogenic genes, including SREBP1c, FAS, ACC-1, and SCD-1 . This indicates that ABHD6 inhibition influences lipid metabolism pathways beyond direct endocannabinoid effects.

• Glucose homeostasis improvement: ABHD6 inhibition protected mice from high-fat diet-induced hyperglycemia and hyperinsulinemia, improving both glucose and insulin tolerance . This suggests effects on insulin signaling pathways and glucose metabolism.

• Cholesterol metabolism modification: ABHD6 ASO treatment protected against high-fat diet-induced hypercholesterolemia, reflected as a significant decrease in LDL and a modest increase in HDL levels . This indicates effects on cholesterol homeostasis pathways.

These diverse effects highlight the importance of considering ABHD6 not merely as a 2-AG hydrolase but as an enzyme with multifaceted roles in various signaling networks. When designing experiments targeting ABHD6, researchers should include readouts for these related pathways to fully understand the consequences of ABHD6 modulation.

What evidence supports ABHD6's role in metabolic disorders?

Multiple lines of evidence support ABHD6's involvement in metabolic disorders, positioning it as a promising therapeutic target:

• Hepatic lipid metabolism: ABHD6 knockdown using antisense oligonucleotides (ASOs) significantly reduced high-fat diet-induced accumulation of hepatic triacylglycerol without altering total hepatic levels of diacylglycerols or monoacylglycerols . This reduction affected almost all molecular species of TAG, suggesting ABHD6 as a potential target for non-alcoholic fatty liver disease.

• Glucose homeostasis: ABHD6 ASO treatment protected mice from high-fat diet-induced hyperglycemia and hyperinsulinemia, significantly improving both glucose and insulin tolerance . These findings suggest ABHD6 inhibition could benefit type 2 diabetes management.

• Lipogenic gene expression: ABHD6 knockdown reduced the expression of key lipogenic genes involved in de novo fatty acid synthesis and lipogenesis (SREBP1c, FAS, ACC-1, and SCD-1) in mouse liver, and reduced the in vivo rate of de novo fatty acid synthesis . This indicates ABHD6 modulates fundamental metabolic pathways controlling lipid synthesis.

• Cholesterol metabolism: ABHD6 ASO treatment protected against high-fat diet-induced hypercholesterolemia, reflected as a significant decrease in LDL and a modest increase in HDL levels . This suggests potential applications in dyslipidemia and cardiovascular disease prevention.

These findings collectively suggest that ABHD6 inhibitors may serve as novel therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes . The metabolic effects appear to be mediated through multiple pathways, making ABHD6 a particularly interesting target for metabolic syndrome, where multiple aspects of metabolism are dysregulated.

How does ABHD6 inhibition affect motor function in neurological disorder models?

Emerging evidence suggests that ABHD6 inhibition may have beneficial effects on motor function in certain neurological disorder models, particularly in a sex-dependent manner:

• Motor performance improvement: Preliminary studies indicate that ABHD6 inhibition improves motor performance specifically in female HdhQ200/200 mice, which serve as a model for Huntington's disease . This sex-dependent effect suggests that ABHD6-targeted therapies might have differential effects based on biological sex.

• Dose-dependent effects: Research has shown that KT-182, a selective ABHD6 inhibitor, at a dose of 2mg/kg can inhibit brain ABHD6 activity by more than 90% when injected intraperitoneally . This high level of target engagement appears necessary for observing the motor performance improvements.

• Administration protocols: Both acute and semi-chronic administration protocols have been utilized to study ABHD6 inhibition effects. For acute studies, animals were monitored for 1 hour after treatment, with behavioral tests conducted 4 hours post-injection to allow for full inhibition of ABHD6 . For semi-chronic studies, researchers have employed Alzet miniature osmotic pumps over daily injections to reduce stress and risk of infection, with drug administered at a rate of 0.5 μl/hour to achieve 2mg/kg/day .

These findings highlight the potential of ABHD6 inhibition as a therapeutic strategy for motor disorders, while also emphasizing the importance of considering sex as a biological variable in both preclinical and clinical studies of ABHD6-targeted interventions. Future research should further characterize the mechanisms underlying these motor effects and explore applications in other neurological conditions involving motor dysfunction.

What methodological considerations are critical when evaluating ABHD6 inhibitors in disease models?

Evaluating ABHD6 inhibitors in disease models requires careful methodological considerations to ensure reliable and translatable results:

• Inhibitor selectivity verification: Given the similarity of ABHD6 to other serine hydrolases, confirming inhibitor selectivity is crucial. Researchers should employ activity-based protein profiling or other techniques to verify that their inhibitor targets ABHD6 without significant off-target effects on related enzymes like MAGL or FAAH. WWL70 has been established as a selective ABHD6 inhibitor that does not significantly affect MAGL activity .

• Dose optimization: Determining the optimal dose for sufficient target engagement is essential. Studies have shown that a dose of 2mg/kg KT-182 inhibits brain ABHD6 activity by more than 90% . Dose-response relationships should be established in each disease model and for each route of administration.

• Temporal considerations: Both acute and chronic effects of ABHD6 inhibition should be assessed, as compensatory mechanisms may develop with long-term inhibition. For acute studies, behavioral tests are typically conducted several hours post-treatment to allow for full ABHD6 inhibition .

• Sex as a biological variable: Evidence suggests that ABHD6 inhibition may have sex-dependent effects, particularly in neurological models . Therefore, both male and female animals should be included in studies, with data analyzed separately by sex before pooling if appropriate.

• Comprehensive phenotyping: Given ABHD6's involvement in multiple physiological systems, studies should include readouts from various domains including metabolic parameters, neurological function, and inflammatory markers to capture the full spectrum of effects.

• Administration method selection: For chronic studies, methods like osmotic mini-pumps may be preferable to daily injections to reduce stress and handling-related variables . The choice of administration method should be justified based on the specific research question and model.

Attention to these methodological details will enhance the rigor and reproducibility of ABHD6 inhibitor studies, facilitating more effective translation to clinical applications.

How can researchers optimize ABHD6 detection in tissues with low expression levels?

Detecting ABHD6 in tissues with low expression levels requires optimized protocols and signal amplification strategies:

• Signal amplification techniques: For immunohistochemistry or immunofluorescence applications, tyramide signal amplification (TSA) can significantly enhance detection sensitivity of HRP-conjugated antibodies by depositing multiple tyramide molecules at the binding site. Similarly, polymer-based detection systems can increase the number of HRP molecules per binding event, boosting signal output.

• Sample preparation refinements: Optimizing antigen retrieval methods is essential for unmasking ABHD6 epitopes, particularly in fixed tissues. Heat-induced epitope retrieval using citrate or EDTA buffers should be systematically compared to determine optimal conditions for ABHD6 detection. Additionally, testing different fixation protocols can help preserve ABHD6 epitopes while maintaining tissue morphology.

• Detection protocol optimization: Extended primary antibody incubation (overnight at 4°C) can increase antibody binding in low-expression samples. For Western blotting applications, selecting highly sensitive enhanced chemiluminescent (ECL) substrates appropriate for low-abundance targets is crucial. Blocking optimization with different agents (BSA, milk, commercial blockers) can help reduce background while maintaining specific signal.

• Concentration techniques: For biochemical analyses, subcellular fractionation to enrich membrane fractions where ABHD6 is localized can enhance detection. Immunoprecipitation to concentrate ABHD6 protein prior to analysis may also improve detection in samples with very low expression.

These approaches should be systematically tested and optimized for each specific tissue type and experimental question, with careful documentation of optimization parameters to ensure reproducibility.

What are the challenges in developing highly selective inhibitors for ABHD6?

Developing selective ABHD6 inhibitors faces several challenges due to the similarity of ABHD6 to other serine hydrolases:

• Structural conservation in the catalytic domain: Many serine hydrolases, including ABHD6, share the α/β-hydrolase fold with a conserved catalytic triad (Ser-His-Asp/Glu). This structural similarity makes it challenging to develop inhibitors that bind selectively to ABHD6 without affecting related enzymes.

• Off-target activity assessment: Traditional enzyme assays may miss off-target effects on less-studied hydrolases. Comprehensive selectivity profiling using activity-based protein profiling (ABPP) with broad-spectrum serine hydrolase probes is essential to assess selectivity across the entire serine hydrolase family.

• Species differences: ABHD6 structure and function may vary between species, affecting inhibitor selectivity in animal models versus humans. Comparative studies of inhibitor selectivity across species are necessary, particularly during preclinical development.

• Tissue-specific considerations: The requirements for inhibitor selectivity may differ across tissues based on the co-expression of related hydrolases. A compound may show acceptable selectivity in brain tissue but have problematic off-target effects in peripheral tissues.

Despite these challenges, progress has been made with selective ABHD6 inhibitors like WWL70, which has been shown to inhibit [³H]-2-AG hydrolysis in ABHD6-expressing systems without significantly affecting MAGL activity . Similarly, KT-182 has demonstrated high potency for ABHD6 inhibition in vivo . Continued refinement of these compounds and development of new chemical scaffolds with improved selectivity profiles remains an active area of research.

How can researchers effectively use ABHD6 antibodies to investigate enzyme-substrate interactions?

Investigating ABHD6's enzyme-substrate interactions requires sophisticated approaches combining antibody-based detection with functional assays:

• Activity-based protein profiling (ABPP): This technique uses active-site directed probes to label catalytically active ABHD6. When combined with ABHD6 antibodies for subsequent detection or immunoprecipitation, ABPP can provide insights into which pool of ABHD6 is catalytically active under various conditions or treatments.

• Substrate trapping approaches: Using catalytically inactive ABHD6 mutants that bind but do not hydrolyze substrates, followed by immunoprecipitation with ABHD6 antibodies and mass spectrometry, can identify physiological substrates. This approach can reveal whether ABHD6 interacts with lipids beyond the established 2-AG substrate.

• Proximity labeling techniques: Fusing promiscuous labeling enzymes like BioID or APEX2 to ABHD6, followed by detection with ABHD6 antibodies, can identify proteins and potentially substrates that come into close proximity with ABHD6 in living cells.

• Co-localization with substrate sensors: Using ABHD6 antibodies in conjunction with fluorescent sensors for lipids like 2-AG can reveal spatial relationships between the enzyme and its substrates at subcellular resolution.

• Structure-function analysis: Combining site-directed mutagenesis of ABHD6 with antibody detection can identify residues critical for substrate binding versus catalysis, providing insights into the molecular basis of ABHD6 substrate selectivity.

These techniques move beyond simple detection of ABHD6 protein and toward understanding its functional interactions with substrates in physiologically relevant contexts, advancing our understanding of ABHD6 biology and potentially revealing novel substrates beyond established monoacylglycerols.

How might ABHD6 research contribute to precision medicine approaches?

ABHD6 research has significant potential to contribute to precision medicine approaches across multiple disease areas:

• Biomarker development: ABHD6 expression, activity levels, or genetic variants may serve as biomarkers for predicting disease susceptibility, progression, or treatment response. For instance, ABHD6 expression patterns could potentially stratify metabolic disease patients who might respond better to ABHD6-targeting therapies.

• Pharmacogenomic applications: Genetic variations in ABHD6 or related genes may influence individual responses to ABHD6 inhibitors or modulators. Identifying these genetic determinants could guide personalized dosing or treatment selection.

• Sex-specific therapeutic approaches: The observed sex-dependent effects of ABHD6 inhibition in motor function suggest that sex should be considered as a biological variable in ABHD6-targeted therapies. This aligns with precision medicine's goal of moving beyond one-size-fits-all approaches.

• Tissue-selective targeting: Given ABHD6's differential expression and functions across tissues, developing tissue-targeted ABHD6 modulators could provide precision in addressing specific disease manifestations while minimizing off-target effects.

• Combination therapy optimization: Understanding how ABHD6 modulation affects multiple signaling pathways can inform rational combination therapies tailored to individual patient needs, particularly in complex conditions like metabolic syndrome or neurodegenerative disorders with multiple underlying pathologies.

As research advances, integrating ABHD6 biology with patient-specific factors could contribute to more personalized and effective therapeutic strategies for metabolic, neurological, and potentially other disorders where ABHD6 plays a role.

What emerging technologies could enhance the study of ABHD6 function and regulation?

Several emerging technologies hold promise for advancing ABHD6 research:

• CRISPR-based approaches: CRISPR-Cas9 gene editing enables precise manipulation of ABHD6 in various model systems. CRISPR activation (CRISPRa) and interference (CRISPRi) technologies allow for temporal control of ABHD6 expression without permanent genetic changes, while base editing can introduce specific mutations to study structure-function relationships.

• Single-cell technologies: Single-cell RNA sequencing and proteomics can reveal cell type-specific expression patterns of ABHD6 and co-regulated genes, providing insights into its regulation within complex tissues. This is particularly relevant given ABHD6's differential expression between neurons and glia .

• Advanced imaging techniques: Super-resolution microscopy combined with ABHD6 antibodies can visualize ABHD6's subcellular localization with unprecedented detail, while techniques like expansion microscopy can enhance visualization of ABHD6 in tissue contexts.

• Chemogenetic and optogenetic tools: Developing ABHD6 variants that can be controlled with light or designer drugs would allow for spatiotemporally precise manipulation of ABHD6 activity in vivo.

• Organoid and microphysiological systems: Studying ABHD6 in human-derived organoids or organ-on-chip platforms could bridge the gap between animal models and human physiology, enhancing translational relevance.

• Computational approaches: Machine learning algorithms applied to large datasets could identify novel relationships between ABHD6 and disease pathways, while molecular dynamics simulations could provide insights into ABHD6's structural flexibility and substrate interactions.

Integration of these technologies into ABHD6 research programs will likely accelerate discovery and translation, providing deeper insights into ABHD6's biological roles and therapeutic potential.