ABHD10 Antibody

What is ABHD10 and why is it important for research?

ABHD10 (Abhydrolase Domain Containing 10) is a mitochondrially-localized enzyme that functions as a hydrolase in human tissues, particularly liver cells. The significance of ABHD10 stems from its involvement in critical biochemical processes, including the deglucuronidation of mycophenolic acid acyl-glucuronide (AcMPAG) . This protein has gained research interest because it regulates the S-palmitoylation status of the nucleophilic active site residue of peroxiredoxin-5 (PRDX5), suggesting a role in redox regulation . Furthermore, ABHD10 may influence mycophenolic acid-induced immunotoxicity, which has clinical implications for patients receiving mycophenolic acid as an immunosuppressant .

Methodologically, researchers investigating mitochondrial function, drug metabolism pathways, or oxidative stress responses would benefit from studying ABHD10. The protein's enzymatic activity makes it a potential target for therapeutic interventions in related metabolic disorders.

What applications can ABHD10 antibodies be used for in research?

ABHD10 antibodies have been validated for multiple research applications, with varying protocols and optimization requirements:

For optimal results across all applications, researchers should titrate antibody concentrations based on their specific sample types. The antibody selection should be guided by the intended application, with consideration given to polyclonal antibodies for broader epitope recognition or when studying potentially modified forms of ABHD10 .

What is the recommended protocol for Western blot detection of ABHD10?

For successful Western blot detection of ABHD10, follow this methodological approach:

• Sample Preparation: Prepare cell or tissue lysates in appropriate lysis buffer. ABHD10 has been successfully detected in Y79 cells and HeLa cells .

• Protein Loading: Load 20-40 μg of protein per lane, as demonstrated in HLM and HLC samples (40 μg was sufficient for detection) .

• Gel Electrophoresis: Use 10% polyacrylamide gels for optimal separation .

• Transfer: Electrotransfer proteins onto PVDF membranes (such as Immobilon-P) .

• Blocking: Block membranes with appropriate blocking buffer (typically 5% non-fat milk or BSA in TBST).

• Primary Antibody Incubation: Dilute ABHD10 antibody to 1:1000-1:6000 in blocking buffer and incubate overnight at 4°C .

• Detection: Use fluorescent dye-conjugated secondary antibodies for quantitative analysis. An Odyssey infrared imaging system has been successfully employed for detection .

• Expected Results: Look for a band at approximately 28 kDa, which is the observed molecular weight for ABHD10, though the calculated molecular weight is 34 kDa .

Troubleshooting tip: If background is high, increase washing steps or adjust antibody dilution. For weak signals, increase protein loading or primary antibody concentration.

How should ABHD10 antibodies be stored and handled for optimal performance?

Proper storage and handling of ABHD10 antibodies is critical for maintaining their reactivity and specificity:

• Storage Temperature: Store at -20°C as recommended by multiple manufacturers. Most ABHD10 antibodies are stable for one year after shipment when stored properly .

• Formulation: Most commercially available ABHD10 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 or similar formulations .

• Aliquoting: For antibodies in liquid form, aliquoting is generally not necessary for -20°C storage, but may be beneficial for frequently used antibodies to minimize freeze-thaw cycles .

• Reconstitution: For lyophilized antibodies, reconstitute in 100 μl of sterile distilled H₂O with 50% glycerol as specified by manufacturers .

• Freeze-Thaw Cycles: Minimize freeze-thaw cycles to preserve antibody function. Multiple manufacturers explicitly warn to avoid repeated freeze/thaw cycles .

• Working Dilutions: Prepare working dilutions immediately before use and discard any unused diluted antibody.

• Safety Considerations: Note that some ABHD10 antibody preparations contain sodium azide (NaN₃), which requires appropriate handling precautions .

How can I validate the specificity of an ABHD10 antibody for my experimental system?

Validating antibody specificity is crucial for reliable research outcomes. For ABHD10 antibodies, consider implementing these methodological approaches:

• Positive Controls: Use cell lines with confirmed ABHD10 expression such as Y79 or HeLa cells, which have been validated in Western blot applications .

• Overexpression Systems: Employ recombinant expression systems like the Bac-to-Bac Baculovirus Expression System in Sf9 cells that has been successfully used for ABHD10 expression . Compare antibody reactivity between mock-transfected and ABHD10-overexpressing cells.

• Knockout/Knockdown Validation: Implement CRISPR/Cas9 knockout or siRNA knockdown of ABHD10 and verify antibody specificity by the absence of signal in these conditions.

• Immunoprecipitation followed by Mass Spectrometry: Perform immunoprecipitation with the ABHD10 antibody followed by mass spectrometry analysis to confirm that the precipitated protein is indeed ABHD10.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (if available, such as the synthesized peptide derived from human ABHD10 corresponding to amino acid residues A93-D143 ) and observe signal reduction in subsequent assays.

• Cross-Reactivity Assessment: If working with non-human samples, evaluate cross-reactivity with the appropriate species. Some ABHD10 antibodies are predicted to react with mouse and rat ABHD10 , but experimental validation is recommended.

• Application-Specific Validation: For each application (WB, IHC, ICC), perform specific validation steps, as antibody performance can vary significantly between applications.

What are the experimental considerations when studying ABHD10's role in deglucuronidation of mycophenolic acid acyl-glucuronide?

Investigating ABHD10's enzymatic function requires careful experimental design and specific methodological approaches:

• Enzyme Source Preparation:

  • For recombinant ABHD10, consider using Sf9 insect cell expression systems as demonstrated in previous research .

  • For human tissue samples, human liver microsomes (HLM), human liver cytosol (HLC), or human liver homogenates (HLH) have been successfully employed .

• Substrate Preparation:

  • Use pure AcMPAG as the substrate. The kinetic parameters determined for ABHD10 include a Km value of 100.7 ± 10.2 μM, providing a guideline for substrate concentration ranges .

• Reaction Conditions:

  • Optimize buffer composition, pH, temperature, and reaction time based on previous research.

  • Consider the effect of cofactors or metal ions, as certain ions (AgNO₃, CdCl₂, CuCl₂) have been shown to inhibit ABHD10 activity .

• Inhibitor Studies:

  • Include known inhibitors such as PMSF, bis-p-nitrophenylphosphate, and DTNB to confirm ABHD10 activity .

  • Note that PMSF has been shown to increase AcMPAG formation from MPA by 1.8-fold in human liver homogenates, providing a useful experimental control .

• Detection Methods:

  • Develop or adopt appropriate assays to measure deglucuronidation activity, such as HPLC or LC-MS/MS methods.

  • Consider using Western blot analysis to correlate ABHD10 protein expression with enzymatic activity .

• Experimental Controls:

  • Include enzyme blanks, substrate blanks, and inhibitor controls.

  • Use known UGT2B7 substrates and inhibitors as reference points, as UGT2B7 catalyzes the formation of AcMPAG from MPA .

How can I optimize immunohistochemical detection of ABHD10 in tissue samples?

For successful immunohistochemical analysis of ABHD10 in tissue samples, follow these methodological recommendations:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin for optimal preservation of ABHD10 antigenic sites.

  • Process and embed in paraffin following standard protocols.

  • Section tissues at 4-6 μm thickness for optimal antibody penetration.

• Antigen Retrieval:

  • Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Optimize the retrieval method empirically as this can significantly impact antibody binding.

  • Test both pressure cooker and microwave-based retrieval methods to determine which provides optimal results.

• Blocking and Antibody Incubation:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide.

  • Use appropriate blocking serum to reduce non-specific binding.

  • Dilute ABHD10 antibody at 1:100-1:200 as recommended . Titrate the antibody to determine optimal dilution for your specific tissue.

  • Incubate primary antibody at 4°C overnight for maximum sensitivity.

• Detection System:

  • Use sensitive detection systems like polymer-based HRP systems for better signal-to-noise ratio.

  • For dual or multi-staining, consider fluorescent-based detection systems to study co-localization with other mitochondrial markers.

• Controls:

  • Include positive control tissues with known ABHD10 expression.

  • Use negative controls by omitting primary antibody or using isotype control.

  • Consider using tissues from knockout models if available as specificity controls.

• Counterstaining and Mounting:

  • Counterstain with hematoxylin for nuclear visualization.

  • Use appropriate mounting media depending on whether aqueous or permanent mounting is preferred.

• Analysis:

  • Evaluate staining pattern with attention to mitochondrial localization, as ABHD10 is a mitochondrial protein .

  • Quantify expression levels using appropriate image analysis software if quantitative assessment is needed.

What are the technical considerations when investigating ABHD10's interaction with peroxiredoxin-5 (PRDX5)?

Investigating protein-protein interactions between ABHD10 and PRDX5 requires specialized techniques and careful experimental design:

• Co-immunoprecipitation (Co-IP):

  • Use ABHD10 antibodies for immunoprecipitation, followed by Western blotting for PRDX5.

  • Consider appropriate lysis conditions that preserve protein-protein interactions while efficiently extracting mitochondrial proteins.

  • Use gentle detergents like CHAPS or digitonin that are less likely to disrupt protein-protein interactions.

  • Include appropriate controls including IgG control, input control, and reverse Co-IP (immunoprecipitate with PRDX5 antibody and detect ABHD10).

• Proximity Ligation Assay (PLA):

  • Utilize PLA to visualize and quantify ABHD10-PRDX5 interactions in situ with subcellular resolution.

  • Optimize antibody dilutions specifically for PLA, which typically requires higher concentrations than standard immunofluorescence.

• FRET/BRET Analysis:

  • Generate fluorescent or bioluminescent fusion proteins of ABHD10 and PRDX5.

  • Analyze energy transfer as an indicator of protein proximity and interaction.

  • Consider the impact of fusion proteins on protein localization and function.

• Functional Assays:

  • Assess S-palmitoylation status of PRDX5 in the presence and absence of ABHD10 using:

    • Acyl-biotin exchange (ABE) assay

    • Click chemistry with alkyne-palmitate

    • Mass spectrometry-based approaches

  • Correlate changes in palmitoylation with alterations in PRDX5's antioxidant activity.

• Mutagenesis Studies:

  • Create targeted mutations in ABHD10's catalytic residues to determine their importance in the regulation of PRDX5 palmitoylation.

  • Generate PRDX5 mutants with altered palmitoylation sites to validate the specific residues regulated by ABHD10.

• Subcellular Localization:

  • Use confocal microscopy with appropriate mitochondrial markers to confirm co-localization of ABHD10 and PRDX5.

  • Consider super-resolution microscopy techniques for more detailed analysis of spatial relationships.

What are common pitfalls when using ABHD10 antibodies and how can they be addressed?

Researchers often encounter these challenges when working with ABHD10 antibodies:

• Non-specific Binding:

  • Problem: Multiple bands observed in Western blot or non-specific staining in IHC/ICC.

  • Solution: Optimize antibody dilution (start with manufacturer recommendations such as 1:1000-1:6000 for WB ); increase washing steps; use more stringent blocking conditions; validate antibody specificity using positive and negative controls.

• Weak or Absent Signal:

  • Problem: No detection or weak detection of ABHD10 despite expected expression.

  • Solution: Increase protein loading (40 μg has been successful for HLM and HLC ); reduce antibody dilution; optimize antigen retrieval methods for IHC; extend primary antibody incubation time; use more sensitive detection systems.

• Discrepancy Between Predicted and Observed Molecular Weight:

  • Problem: ABHD10 has a calculated molecular weight of 34 kDa but is often observed at 28 kDa .

  • Solution: This is a known characteristic of ABHD10 and likely reflects post-translational modifications or proteolytic processing. Use positive controls to confirm the correct band.

• Inconsistent Results Between Experiments:

  • Problem: Variable detection of ABHD10 across experimental replicates.

  • Solution: Standardize protein extraction methods; use fresh antibody aliquots; maintain consistent experimental conditions; implement quantitative loading controls.

• Cross-Reactivity Issues:

  • Problem: When working with non-human samples, uncertainty about antibody specificity.

  • Solution: Verify cross-reactivity experimentally even if predicted to react with mouse and rat ABHD10 ; consider species-specific antibodies for critical experiments.

• Storage-Related Degradation:

  • Problem: Reduced antibody performance over time.

  • Solution: Adhere to storage recommendations (-20°C ); avoid repeated freeze-thaw cycles; aliquot antibodies for single use; check expiration dates.

How can I distinguish between different isoforms of ABHD10 in my experiments?

ABHD10 has three known isoforms , which presents challenges for isoform-specific detection and analysis:

• Antibody Selection:

  • Determine the epitope region recognized by your antibody. The Invitrogen antibody, for example, targets a synthesized peptide derived from human ABHD10 corresponding to amino acid residues A93-D143 .

  • Verify whether this epitope is present in all isoforms or is isoform-specific by aligning sequences.

  • Consider generating or sourcing isoform-specific antibodies if commercially available antibodies cannot distinguish between isoforms.

• PCR-Based Methods:

  • Design isoform-specific primers for RT-PCR or qPCR to quantify isoform expression at the mRNA level.

  • Use the full-length human ABHD10 cDNA (accession no. NM_018394.2) as a reference for primer design .

  • Validate primer specificity using cloned isoform standards.

• Western Blot Resolution:

  • Use high-percentage gels (12-15%) or gradient gels to improve separation of closely sized isoforms.

  • Consider using 2D gel electrophoresis to separate isoforms based on both molecular weight and isoelectric point.

  • Optimize electrophoresis conditions for maximum resolution.

• Mass Spectrometry Approaches:

  • Employ targeted proteomics to identify unique peptides specific to each isoform.

  • Use immunoprecipitation followed by mass spectrometry for isoform identification.

  • Quantify relative abundance of isoforms using label-free quantitation or isotope labeling strategies.

• Recombinant Expression:

  • Generate recombinant expression constructs for each isoform with epitope tags.

  • Use these as positive controls and for antibody validation.

  • The pFastBac1 vector system has been successfully used for ABHD10 expression and could be adapted for isoform expression .

• Functional Analysis:

  • Assess potential functional differences between isoforms using enzymatic assays.

  • Determine isoform-specific subcellular localization patterns using tagged constructs.

  • Investigate isoform-specific protein-protein interactions.

How can ABHD10 antibodies be used in studies investigating mycophenolic acid-induced immunotoxicity?

Mycophenolic acid (MPA) immunotoxicity is associated with its metabolite AcMPAG, which is deglucuronidated by ABHD10 . Researchers can employ ABHD10 antibodies in the following methodological approaches:

• Expression Analysis in Patient Samples:

  • Use immunohistochemistry with ABHD10 antibodies (dilution 1:100-1:200 ) on liver biopsies from patients on MPA therapy.

  • Correlate ABHD10 expression levels with clinical measures of immunotoxicity.

  • Compare expression patterns between patients with and without adverse reactions.

• Pharmacogenetic Studies:

  • Combine genotyping of ABHD10 variants with protein expression analysis using Western blot (dilution 1:1000-1:6000 ).

  • Investigate whether genetic polymorphisms affecting ABHD10 expression or function correlate with altered susceptibility to MPA toxicity.

• Cell-Based Assays:

  • Develop in vitro models with variable ABHD10 expression levels.

  • Use ABHD10 antibodies to confirm protein levels following genetic manipulation (overexpression, knockdown, or knockout).

  • Assess the impact of ABHD10 levels on AcMPAG accumulation and cellular toxicity markers.

• Mechanistic Studies:

  • Employ ABHD10 antibodies in combination with inhibitor studies (e.g., using PMSF which increases AcMPAG formation 1.8-fold ).

  • Investigate the regulatory mechanisms controlling ABHD10 expression and activity under inflammatory conditions.

  • Explore potential compensatory mechanisms when ABHD10 activity is compromised.

• Biomarker Development:

  • Evaluate ABHD10 as a potential biomarker for predicting MPA toxicity.

  • Develop quantitative assays using antibody-based approaches such as ELISA (dilution 1:20000-1:80000 ).

  • Correlate ABHD10 levels or activity with drug metabolism parameters and clinical outcomes.

What are emerging techniques for studying ABHD10's role in mitochondrial function?

As a mitochondrial protein , ABHD10's role in mitochondrial biology can be investigated using these advanced methodological approaches:

• Live Cell Imaging:

  • Use fluorescently tagged ABHD10 constructs combined with mitochondrial markers to visualize dynamic localization in living cells.

  • Employ super-resolution microscopy techniques (STED, PALM, STORM) for nanoscale localization within mitochondrial compartments.

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to study ABHD10 mobility within mitochondria.

• Mitochondrial Isolation and Proteomic Analysis:

  • Perform subcellular fractionation to isolate pure mitochondrial preparations.

  • Use ABHD10 antibodies to confirm enrichment in mitochondrial fractions via Western blot.

  • Combine with proximity labeling approaches (BioID, APEX) to identify the ABHD10 interaction network within mitochondria.

• Functional Mitochondrial Assays:

  • Measure the impact of ABHD10 modulation on:

    • Mitochondrial membrane potential

    • Oxygen consumption rate

    • ATP production

    • Reactive oxygen species generation

  • Correlate ABHD10 expression (quantified using validated antibodies) with these functional parameters.

• Mitochondrial Stress Responses:

  • Investigate ABHD10's role during mitochondrial stress using:

    • Chemical stressors (rotenone, antimycin A)

    • Hypoxia/reoxygenation models

    • mtDNA depletion approaches

  • Monitor changes in ABHD10 localization, expression, and post-translational modifications.

• CRISPR-Based Approaches:

  • Generate ABHD10 knockout cell lines using CRISPR/Cas9

  • Create knock-in models with tagged or mutant ABHD10

  • Implement CRISPRa/CRISPRi for conditional modulation of ABHD10 expression

  • Validate all genetic manipulations using ABHD10 antibodies

• In Vivo Mitochondrial Studies:

  • Develop animal models with tissue-specific ABHD10 manipulation

  • Analyze mitochondrial morphology, number, and function in these models

  • Use immunohistochemistry with ABHD10 antibodies to correlate protein expression with phenotypic outcomes

How should I select between different commercial ABHD10 antibodies for my specific research application?

Selecting the optimal ABHD10 antibody requires systematic evaluation based on your specific experimental needs:

When selecting an antibody, consider:

• Research Question Alignment:

  • For basic detection of ABHD10, any validated antibody may be suitable

  • For isoform-specific studies, select antibodies with epitopes in unique regions

  • For cross-species studies, choose antibodies with predicted or validated cross-reactivity

• Technical Requirements:

  • Application compatibility (some antibodies are validated for specific applications)

  • Species reactivity needs

  • Sensitivity requirements (some applications like ELISA require higher dilutions)

• Experimental Validation:

  • Review available validation data from manufacturers

  • Consider testing multiple antibodies in pilot experiments

  • Implement appropriate controls to confirm specificity

• Practical Considerations:

  • Storage requirements and stability

  • Format (liquid vs. lyophilized)

  • Cost and availability

What experimental design would best evaluate ABHD10's potential as a therapeutic target?

A comprehensive evaluation of ABHD10 as a therapeutic target would require a multi-faceted experimental approach:

• Target Validation Phase:

  • Expression Analysis:

    • Quantify ABHD10 expression across tissues and disease states using validated antibodies

    • Compare expression in normal vs. pathological samples (e.g., tissues with altered drug metabolism)

    • Perform subcellular localization studies to confirm mitochondrial targeting

  • Function-Phenotype Correlation:

    • Generate ABHD10 knockout models (cellular and animal)

    • Assess phenotypic consequences of ABHD10 deletion or inhibition

    • Evaluate compensatory mechanisms that might affect therapeutic outcomes

• Inhibitor Development and Evaluation:

  • High-Throughput Screening:

    • Develop enzyme activity assays suitable for screening

    • Screen compound libraries for ABHD10 inhibitors

    • Validate hits using secondary assays including Western blot to confirm target engagement

  • Structure-Activity Relationship (SAR) Studies:

    • Generate a structural model of ABHD10 to guide inhibitor design

    • Synthesize analogs of initial hits to improve potency and selectivity

    • Use antibody-based assays to confirm binding to ABHD10 protein

• Efficacy Assessment:

  • Cellular Models:

    • Test inhibitors in cellular systems with validated ABHD10 expression

    • Measure impact on AcMPAG metabolism and related toxicity

    • Assess effects on mitochondrial function and redox regulation

  • Animal Models:

    • Develop appropriate animal models for testing

    • Evaluate pharmacokinetics and pharmacodynamics of inhibitors

    • Assess efficacy in disease-relevant endpoints

• Biomarker Development:

  • Identify biomarkers of ABHD10 inhibition using proteomics and metabolomics

  • Develop antibody-based assays for monitoring target engagement

  • Establish correlation between biomarker changes and functional outcomes

• Safety Assessment:

  • Evaluate potential off-target effects

  • Assess impact on mitochondrial function across multiple tissues

  • Determine potential drug-drug interactions, particularly with medications metabolized through glucuronidation pathways

• Translational Studies:

  • Correlate findings from preclinical models with human data

  • Investigate ABHD10 expression in relevant patient populations

  • Develop clinical trial protocols with appropriate patient stratification strategies