ACAD10 Antibody

What is ACAD10 and why is it studied in research?

ACAD10 (Acyl-CoA dehydrogenase family member 10) is a member of the acyl-CoA dehydrogenase family of enzymes that participate in the beta-oxidation of fatty acids in mitochondria. The protein contains multiple domains: a hydrolase domain at the N-terminal portion, a serine/threonine protein kinase catalytic domain in the central region, and a conserved ACAD domain at the C-terminus . ACAD10 has gained significant research interest due to its association with type 2 diabetes in certain populations, particularly in Native American Pima Indians who have the highest reported incidence of insulin resistance and type 2 diabetes mellitus . Additionally, ACAD10 has significant activity towards branched-chain substrates R and S, 2-methyl-C15-CoA and shows a unique expression pattern with high levels in fetal brain but not adult brain .

What types of ACAD10 antibodies are available for research?

There are multiple types of ACAD10 antibodies available, including:

• Polyclonal antibodies:

  • Examples include products like the Proteintech 17161-1-AP, which targets ACAD10 in Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF)/ICC, and ELISA applications with reactivity to human, mouse, and rat samples .

  • The Atlas Antibodies polyclonal antibody against human ACAD10 (HPA067222) .

• Monoclonal antibodies:

  • Recombinant rabbit monoclonal antibodies like the ZooMAb® 3J20 clone that specifically detects ACAD10 with high specificity across multiple applications .

The choice between polyclonal and monoclonal depends on the specific research application, with monoclonals typically offering higher specificity but potentially lower sensitivity compared to polyclonals.

What is the molecular weight of ACAD10 protein in Western blot analysis?

• The calculated molecular weight can appear as both 26 kDa and 119 kDa, depending on the isoform detected .

• Recent research has demonstrated that ACAD10 undergoes cleavage in cells, resulting in separate N-terminal and C-terminal fragments that can be detected at different molecular weights .

• Proteomic analysis has identified that some antibodies may detect both the full-length ~119 kDa band of ACAD10 and a ~45 kDa band that corresponds to the mitochondrial isoform .

When validating ACAD10 antibodies, it's critical to confirm the expected banding pattern using positive controls and knockout samples if available.

What are the recommended dilutions for ACAD10 antibodies in different applications?

Based on validated antibody data, the following dilutions are typically recommended:

What tissues show positive detection of ACAD10 with antibodies?

ACAD10 antibodies have shown positive detection in several tissues:

• Western Blot: Positive detection in mouse spleen tissue

• Immunohistochemistry: Positive detection in human pancreas tissue (using antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0)

• Immunofluorescence/ICC: Positive detection in HeLa cells

Additionally, ACAD10 antigen has been detected in multiple mouse tissues including lung, muscle, kidney, and pancreas, with localization primarily to mitochondria and weak signals in peroxisomes of mouse lung . In human tissues, ACAD10 has been detected in lung, kidney, liver, muscle, and pancreas, with the antigen present in mitochondria and weak signals in peroxisomes in kidney and pancreas .

How should sample preparation be optimized for ACAD10 detection in immunohistochemistry?

For optimal detection of ACAD10 in immunohistochemistry applications:

• Fixation: Tissues should be harvested and immersed in fixative (typically 4% paraformaldehyde) overnight .

• Antigen retrieval: For paraffin-embedded sections, antigen retrieval is crucial:

  • Primary recommendation: Use TE buffer at pH 9.0

  • Alternative method: Use citrate buffer at pH 6.0

• Antibody incubation:

  • Primary antibody: Incubate overnight at 4°C using dilutions between 1:20-1:200

  • Secondary antibody: Incubate for 1 hour with fluorescently labeled secondary antibodies

• Counterstaining: Use appropriate counterstains based on the detection method (fluorescent or chromogenic)

For subcellular localization studies, co-staining with mitochondrial markers (such as anti-MTCO1 antibody) and peroxisomal markers (such as catalase) can help confirm the organelle-specific localization of ACAD10 .

How can researchers distinguish between different isoforms of ACAD10 in experimental systems?

Distinguishing between different ACAD10 isoforms requires careful experimental design:

• Using domain-specific antibodies: Recent research has identified that ACAD10 is cleaved into two parts in cells . Researchers can use antibodies targeting different domains (HAD domain, kinase domain, or ACAD domain) to distinguish between isoforms.

• Molecular weight analysis: The full-length ACAD10 appears at ~119 kDa, while a mitochondrial isoform may appear at ~45 kDa . Careful analysis of band patterns can help identify specific isoforms.

• Subcellular fractionation: Isolating mitochondrial, peroxisomal, and cytosolic fractions can help distinguish isoforms based on their localization. The ACAD10 antigen-purified antibody has identified the predicted 119 kDa ACAD10 protein in mitochondrial/peroxisomal fractions, plus a smaller sized protein in purified peroxisomal fractions .

• PCR analysis of transcript variants: For a comprehensive analysis, researchers can design primer sets targeting different exons to identify specific transcript variants that may encode different protein isoforms .

How should researchers validate ACAD10 antibody specificity in their experimental systems?

Rigorous validation of ACAD10 antibodies is essential for reliable results:

• Knockout/knockdown controls:

  • Generate ACAD10 knockout cell lines via CRISPR/Cas9 technology

  • Use ACAD10 siRNA knockdown

  • As demonstrated in recent research, comparing wildtype and ACAD10KO samples can confirm specificity

• Proteomic confirmation:

  • Excise bands from Western blots and perform proteomic profiling

  • Confirm peptide identities via mass spectrometry

  • Research has shown this approach can identify ACAD10-specific peptides and distinguish between isoforms

• Multiple antibody approach:

  • Use antibodies from different sources/clones targeting different epitopes

  • Compare staining patterns to ensure consistency

• Rescue experiments:

  • Restore ACAD10 expression in knockout lines using recombinant lentiviruses

  • Test variants with domain-specific mutations to verify antibody epitope specificity

What techniques can address contradictory findings regarding ACAD10 subcellular localization?

Conflicting results regarding ACAD10 subcellular localization can be resolved through:

• Complementary localization techniques:

  • Immunofluorescence with co-localization markers

  • Subcellular fractionation followed by Western blotting

  • Proximity labeling approaches (BioID, APEX)

  • Electron microscopy with immunogold labeling

• Domain-specific targeting:

  • ACAD10 has a predicted N-terminal mitochondrial targeting sequence (MTS)

  • ACAD11 has a predicted C-terminal peroxisomal targeting sequence (PTS)

  • Creating domain-specific fluorescent fusion proteins can help track localization

• Cell-type considerations:

  • Localization may differ between cell types and tissues

  • ACAD10 shows primarily mitochondrial localization in most tissues with weak peroxisomal signals in specific tissues like kidney and pancreas

• Isoform-specific analysis:

  • Different transcripts may encode proteins with different targeting sequences

  • The full-length vs. processed forms may localize differently

What is the current understanding of ACAD10's enzymatic activity and substrates?

ACAD10's enzymatic activity has been characterized in several studies:

• Substrate specificity:

  • ACAD10 shows significant activity towards branched-chain substrates R and S, 2-methyl-C15-CoA

  • Recent research indicates ACAD10 and ACAD11 enable the entry of 4-hydroxy fatty acids into β-oxidation pathways

  • The enzyme requires CoA-conjugated substrates, as neither ACAD10 nor ACAD11 were able to phosphorylate the free acid form of 4-hydroxy valerate (4-HV)

• Multi-domain functionality:

  • The C-terminal ACAD domain is responsible for dehydrogenation activity

  • The N-terminal haloacid dehalogenase (HAD) domain may have phosphatase activity dependent on a DxD motif

  • The central kinase domain contains conserved residues like aspartate 463 that are potentially required for activity

• Organelle-specific function:

  • ACAD10 and ACAD11 may have non-redundant roles in cellular 4-hydroxy acid (4-HA) catabolism based on their distinct subcellular localizations

How does ACAD10 deficiency relate to metabolic disorders in research models?

Research on ACAD10 deficiency and metabolic disorders has yielded complex findings:

• Type 2 diabetes connection:

  • ACAD10 gene polymorphisms have been linked to type 2 diabetes susceptibility in Pima Indians

  • An ACAD10-deficient mouse model on a SvEv129/BL6 mixed background exhibited abnormal glucose tolerance tests, elevated insulin levels, and accumulation of excess abdominal adipose tissue

  • These mice also developed early inflammatory liver processes, exhibited fasting rhabdomyolysis, and had abnormal skeletal muscle mitochondria

• Contradictory findings:

  • A recent study using ACAD10KO mice on a C57BL/6J background found no metabolic phenotype, with no differences in body composition, energy expenditure, or glucose tolerance compared to wildtype mice

  • This study also found that ACAD10 was not required for metformin's metabolic actions, despite previous reports that ACAD10 upregulation was necessary for metformin's effects in other contexts

• Molecular alterations:

  • Despite the lack of a metabolic phenotype in C57BL/6J mice, ACAD10 depletion influenced the abundance of specific ceramide species containing very long chain fatty acids

  • ACAD10 was identified as the top downregulated gene in the kidney of a mouse model of diabetes, and interventions that protected against renal damage restored ACAD10 levels

• Model system considerations:

  • The contradictory findings between mouse models suggest genetic background effects

  • Differences in study design, including use of littermate controls versus separate breeding of lines, may influence outcomes

What methodological approaches can help resolve contradictions in ACAD10 research findings?

To address contradictory findings in ACAD10 research, consider:

• Genetic background standardization:

  • Use mice on pure genetic backgrounds (e.g., C57BL/6J) with appropriate littermate controls

  • When comparing to previous studies, account for background strain differences (e.g., SvEv129/BL6 mixed vs. pure background)

• Experimental design improvements:

  • Increase sample sizes to improve statistical power

  • Implement blinding procedures for phenotyping to minimize bias

  • Use multiple diet conditions (normal chow, high-fat high-sucrose) and examine both sexes

• Comprehensive phenotyping:

  • Perform in-depth metabolic assessments including glucose tolerance tests, body composition analysis, and energy expenditure measurements

  • Analyze mitochondrial function using techniques like high-resolution respirometry

  • Employ lipidomic profiling to identify subtle changes in lipid composition

• Tissue-specific and developmental considerations:

  • Examine multiple tissues (liver, muscle, adipose, kidney, brain)

  • Consider developmental stages, as ACAD10 is highly expressed in fetal brain compared to adult brain

  • Analyze both young and aged animals to capture age-dependent phenotypes

How can ACAD10 antibodies be used to study post-translational modifications and protein processing?

ACAD10 undergoes complex processing that can be studied using domain-specific antibodies:

• Protein cleavage analysis:

  • Recent research has shown that ACAD10 is cleaved in cells, with peptides beyond amino acid 617 essentially absent when purifying the N-terminal part

  • Antibodies targeting different domains can help track the fate of cleaved products

• Mitochondrial processing:

  • The involvement of mitochondrial processing peptidase (MPP) in ACAD10 cleavage can be studied using antibodies against both intact and processed forms

  • Multiple arginine residues (R603, R616, R623, R634) may serve as cleavage sites

• Domain-specific antibodies:

  • Antibodies targeting the HAD domain, kinase domain, or ACAD domain can help understand domain-specific functions and interactions

  • Using epitope-tagged constructs with domain-specific mutations can provide additional insights into processing mechanisms

What are the recommended controls and validation steps for using ACAD10 antibodies in multi-omics research?

For integrating ACAD10 antibody data with multi-omics approaches:

• Orthogonal validation approaches:

  Validation Method	Application	Benefit
  Proteomics	Mass spectrometry identification of immunoprecipitated proteins	Confirms antibody specificity and identifies interacting partners
  Transcriptomics	RNA-seq correlation with protein levels	Validates antibody-detected changes at transcriptional level
  Genomics	Use of CRISPR-modified cells	Provides definitive negative controls
  Metabolomics	Correlation of enzyme levels with substrate/product ratios	Functional validation of antibody-detected changes

• Integrated analysis pipeline:

  • Verify antibody-detected changes in protein levels correlate with transcript abundance

  • Confirm functional consequences through metabolite profiling, particularly of fatty acid intermediates

  • Use phospho-specific antibodies to correlate with phosphoproteomic datasets

• Systems biology approach:

  • Use network analysis to position ACAD10 in relevant pathways

  • Correlate antibody-detected ACAD10 levels with other components of fatty acid metabolism

  • Compare findings across multiple antibodies targeting different epitopes

How can researchers optimize ACAD10 antibodies for studying its role in 4-hydroxy acid lipid metabolism?

To study ACAD10's emerging role in 4-hydroxy acid metabolism:

• Substrate-specific activity assays:

  • Use antibodies to immunoprecipitate ACAD10 for in vitro activity assays with 4-hydroxy-CoA substrates

  • Compare activity between full-length and cleaved forms of the protein

  • Correlate enzyme levels detected by antibodies with metabolic flux through 4-hydroxy acid pathways

• Organelle-specific localization:

  • Optimize subcellular fractionation protocols to separate mitochondria and peroxisomes

  • Use antibodies against different ACAD10 domains to track localization of processed forms

  • Correlate localization with 4-hydroxy acid metabolism in different cellular compartments

• Interaction partners:

  • Use antibodies for co-immunoprecipitation to identify proteins that interact with ACAD10 in the context of 4-hydroxy acid metabolism

  • Verify interactions through reciprocal pulldowns and proximity labeling approaches

  • Determine if interactions differ between full-length and processed forms of ACAD10

• Metabolic tracing studies:

  • Correlate ACAD10 levels detected by antibodies with metabolic flux through 4-hydroxy acid pathways using stable isotope tracing

  • Compare wildtype and CRISPR/Cas9-edited cells to determine ACAD10's contribution to specific metabolic pathways