ACAD9 Antibody

What is ACAD9 and why is it important in mitochondrial research?

ACAD9 (Acyl-CoA Dehydrogenase Family Member 9) is a multifunctional mitochondrial flavoenzyme that plays dual roles in cellular metabolism. It catalyzes the initial rate-limiting α,β-dehydrogenation step in fatty acid β-oxidation while simultaneously serving as an essential assembly factor for mitochondrial respiratory complex I .

ACAD9 is particularly significant because:

• It has a calculated molecular mass of 68.8 kDa (observed at 60-65 kDa on gels)

• It contains an N-terminal leader sequence and two conserved motifs shared by all ACAD family members

• Defects in ACAD9 cause a distinct genetic disorder in mitochondrial fatty acid β-oxidation

• It's the most common cause of isolated complex I deficiency in humans

How does ACAD9 structure compare to other ACAD family members?

ACAD9 shares significant structural homology with VLCAD (Very Long-Chain Acyl-CoA Dehydrogenase):

• 46.4% sequence identity and 77.6% sequence similarity with human VLCAD

• Both exist as homodimers with similar elution profiles on size exclusion chromatography

• Contains MCAD-like N-terminal domain and a C-terminal domain linked by an arginine-rich region

Despite these similarities, ACAD9 demonstrates unique properties:

• Lower binding affinity for FAD compared to VLCAD (~70% FAD occupancy in purified ACAD9 vs. full stoichiometric amount in VLCAD)

• Approximately 10% of VLCAD's fatty acid oxidation activity

• Less stable homodimer interface due to amino acid differences (e.g., Ser395 in ACAD9 vs. Asp391 in VLCAD)

How does interaction with ECSIT affect ACAD9 function and antibody binding?

The interaction between ACAD9 and ECSIT has profound effects on ACAD9's biochemical properties that may impact antibody-based experiments:

• Conformational changes: When ECSIT interacts with ACAD9, the flavoenzyme loses its FAD cofactor and consequently loses its fatty acid oxidation activity

• Binding site: ECSIT binds to ACAD9 via its C-terminal domain (residues 320-334) , which can potentially mask antibody epitopes in this region

• Complex stability: The binary complex of ACAD9 with ECSIT or with C-ECSIT is unstable and aggregates easily, while the ternary complex with NDUFAF1 is highly stable

• Research implications: Researchers should consider these interactions when designing experiments to study either fatty acid oxidation or complex I assembly functions

Recent cryo-EM structural studies revealed that a synthetic peptide spanning ECSIT residues 318-336 was sufficient to cause deflavination of ACAD9 , suggesting careful consideration when selecting antibodies targeting regions near this interaction surface.

What methodological approaches can distinguish between ACAD9's dual functions?

Distinguishing between ACAD9's roles in fatty acid oxidation versus complex I assembly requires specialized experimental approaches:

• Activity-based assays:

  • Fatty acid oxidation activity can be measured using substrates like C16-CoA

  • Complex I assembly can be assessed through Blue Native PAGE and respiratory chain complex activity assays

• Protein interaction studies:

  • Pull-down assays using ACAD9-His6 can identify interactions with ECSIT and NDUFAF1

  • Immunoprecipitation with specific antibodies can isolate different ACAD9-containing complexes

• Strategic antibody selection:

  • Antibodies targeting regions unaffected by ECSIT binding are preferred for detecting total ACAD9

  • Conformation-specific antibodies might differentiate between FAD-bound and FAD-free forms

• FAD occupancy measurement:

  • UV-Vis spectroscopy can determine FAD content in purified ACAD9 protein

  • Higher FAD occupancy correlates with fatty acid oxidation activity

How can researchers validate ACAD9 antibody specificity?

Rigorous validation of ACAD9 antibodies is critical for research applications. Based on published methodologies:

• Genetic models:

  • ACAD9-deficient mouse models show no signal with ACAD9 antibody compared to wild-type littermates

  • Tissue-specific knockouts (cardiac-specific, muscle-specific) provide additional controls

• Immunocompetition assays:

  • Add purified ACAD9 protein to antibody incubation solution at 1:1 molar ratio

  • Observe signal reduction in the presence of competing antigen

• Immunoinactivation:

  • Add purified antibody to liver lysate protein (1 μg antibody per 10 μg protein)

  • Incubate at 4°C overnight, then precipitate with protein A-bound resin

  • Assay supernatant for remaining ACAD9 activity

• Molecular weight verification:

  • ACAD9 appears at 68.8 kDa (calculated) or 60-65 kDa (observed on gels)

  • Secondary bands may represent processed or modified forms

What are the optimal extraction and sample preparation methods for ACAD9 detection?

Based on published protocols, the following methods yield optimal results for ACAD9 antibody-based detection:

• Protein extraction from cells:

  • Total protein from human fibroblasts: Standard lysis buffers with protease inhibitors

  • For membrane-associated ACAD9: Consider mitochondrial isolation followed by membrane solubilization

• Protein extraction from tissues:

  • Liver samples: TRIzol reagent can be used for RNA and protein extraction

  • Muscle samples: 150 μg of total protein is recommended for optimal detection

• Sample preparation for immunoblotting:

  • Separate 50 μg of total protein from fibroblast/liver extracts on 12% SDS polyacrylamide gel

  • Transfer to nitrocellulose for optimal antibody binding

• Preservation of native interactions:

  • For pull-down assays: Use mild detergents and avoid harsh denaturants

  • For native complex detection: Consider blue native PAGE techniques

What are the critical parameters for optimizing ACAD9 antibody-based assays?

Several parameters must be optimized when using ACAD9 antibodies in research applications:

• Western blotting optimization:

  • Recommended dilutions range from 1:500-1:3000 depending on the antibody source

  • Secondary antibody: Typically goat anti-rabbit HRP at 1:3000 dilution

  • Blocking: 1% non-fat dry milk in PBST is commonly used

• Immunohistochemistry considerations:

  • Antigen retrieval: TE buffer pH 9.0 or citrate buffer pH 6.0

  • Dilution ranges: 1:50-1:500 depending on tissue type and fixation method

• Storage and handling:

  • Store antibodies at -20°C or -80°C as recommended by manufacturer

  • Avoid repeated freeze-thaw cycles that may reduce antibody activity

  • For frequent use, aliquot and store at 4°C for up to one month

How can researchers troubleshoot ACAD9 antibody experiments?

Common challenges and troubleshooting approaches for ACAD9 antibody experiments include:

• Low signal intensity:

  • Increase protein loading (up to 150 μg for muscle tissue)

  • Decrease antibody dilution within recommended ranges

  • Enhance detection using signal amplification systems

• Multiple bands or unexpected molecular weight:

  • ACAD9 calculated weight is 68.8 kDa but often appears at 60-65 kDa

  • Multiple bands may represent processing of the N-terminal leader sequence

  • Verify with positive control samples (HEK-293T cells, MCF-7 cells)

• Poor reproducibility:

  • Standardize protein extraction protocols

  • Use freshly prepared samples when possible

  • Include loading controls (Beta Actin, GUSB, or 18S rRNA)

• Background issues:

  • Increase blocking concentration or duration

  • Add 0.1% BSA to storage buffer if needed

  • Consider specialized blocking reagents for tissues with high background

How are ACAD9 antibodies being used to study disease mechanisms?

ACAD9 antibodies have been instrumental in studying several disease mechanisms:

• Genetic disorders of fatty acid oxidation:

  • Identification of three cases of ACAD9 deficiency with Reye-like episodes and liver dysfunction

  • Characterization of ACAD9 protein levels in patient fibroblasts

• Mitochondrial respiratory chain disorders:

  • Investigation of complex I assembly defects in patient samples

  • Mapping of over 40 pathogenic mutation sites onto modeled ACAD9 structure

• Cardiomyopathy models:

  • Cardiac-specific ACAD9 deficient animals develop severe neonatal cardiomyopathy

  • Western blotting confirms absence of ACAD9 protein in affected tissues

• Neurodegenerative diseases:

  • ACAD9 has been studied in relation to Alzheimer's disease pathology

  • Immunofluorescence validation based on peptide sequence homology analysis

What emerging techniques are enhancing ACAD9 antibody applications?

Recent technological advances are expanding the utility of ACAD9 antibodies:

• Cryo-EM structural studies:

  • 3.0 Å resolution cryo-EM structure of ACAD9-ECSIT complex recently solved

  • Helps identify epitope accessibility in different protein conformations

• AlphaFold2 structural predictions:

  • AI-predicted structures of ACAD9-ECSIT complex match experimental structures (RMSD of 1.635 Å)

  • Useful for epitope selection in antibody production

• Mass photometry:

  • Used to analyze ACAD9 dimers and complexes with ECSIT

  • Provides insights into molecular weight and stoichiometry without labels

• Promoter analysis techniques:

  • Luciferase reporter assays used to study ACAD9 promoter function

  • Helps understand regulatory mechanisms affecting ACAD9 expression

What are the most promising therapeutic research avenues utilizing ACAD9 antibodies?

ACAD9 antibodies are supporting several therapeutic research directions:

• Mouse model development:

  • Tissue-specific ACAD9 deficient animals serve as useful models for testing novel therapeutics

  • Cardiac and muscle-specific knockouts provide systems for targeted intervention testing

• Pathway modulation strategies:

  • ECSIT phosphorylation affects ACAD9-ECSIT interaction, offering potential regulatory targets

  • Understanding complex I assembly mechanism may lead to assembly-promoting therapeutics

• Biomarker development:

  • Quantitative assessment of ACAD9 protein levels using antibody-based assays

  • Correlation with disease severity and progression

• Recombinant protein therapies:

  • Antibodies help characterize recombinant ACAD9 properties

  • Potential for enzyme replacement strategies in deficiency conditions