ACADS Antibody

What is ACADS and why is it significant in research?

ACADS, also known as SCAD (Short-chain specific acyl-CoA dehydrogenase, mitochondrial), is an enzyme that catalyzes the first step of mitochondrial fatty acid beta-oxidation . It plays a critical role in the aerobic process of breaking down fatty acids into acetyl-CoA, allowing energy production from fats . ACADS specifically acts on acyl-CoAs with saturated 4 to 6 carbons long primary chains, making it an important enzyme in metabolic research . The enzyme is synthesized as a 44 kDa precursor that is transported into mitochondria and proteolytically processed to its 41 kDa mature form .

What types of ACADS antibodies are available for research?

There are multiple types of ACADS antibodies available for research applications, including:

• Polyclonal antibodies: Such as rabbit polyclonal IgG antibodies that target ACADS in various applications

• Monoclonal antibodies: Including rabbit recombinant monoclonal antibodies specific to ACADS/SCAD

Each antibody type offers different advantages depending on the experimental design and research objectives. Polyclonal antibodies recognize multiple epitopes on the target antigen, while monoclonal antibodies offer higher specificity to a single epitope.

In which applications can ACADS antibodies be used?

ACADS antibodies have been validated for multiple laboratory applications, as shown in the following table:

The optimal dilution may be sample-dependent, so it's recommended to titrate the antibody in each testing system to obtain optimal results .

What species reactivity can be expected from ACADS antibodies?

Most commercially available ACADS antibodies demonstrate reactivity across multiple species. According to validation studies, these antibodies typically show reactivity with:

• Human samples

• Mouse samples

• Rat samples

• Monkey samples

This cross-reactivity makes these antibodies valuable tools for comparative studies across different model organisms.

How should ACADS antibody specificity be validated for critical research applications?

For rigorous validation of ACADS antibody specificity, researchers should employ at least one of the five conceptual pillars of antibody validation recommended by the International Working Group for Antibody Validation :

• Genetic strategies: Eliminate or significantly reduce the expression of ACADS protein by genome editing or RNA interference to confirm antibody specificity . This approach provides definitive evidence that the antibody recognizes the intended target.

• Orthogonal strategies: Compare antibody-based ACADS detection with an antibody-independent method (e.g., targeted proteomics with labeled internal standards) . Strong correlation between these measurements across samples with variable ACADS expression confirms specificity.

• Independent antibody strategies: Use two ACADS antibodies with non-overlapping epitopes and compare their detection patterns . Concordant results strengthen validation.

• Tagged protein expression: Express ACADS with a tag (preferably at endogenous levels) and confirm co-localization of the antibody signal with the tag .

• Immunocapture followed by mass spectrometry (IMS): Capture ACADS using the antibody and analyze the isolated protein by mass spectrometry to confirm identity .

For critical applications, implementing multiple validation strategies significantly strengthens confidence in antibody specificity.

What are the potential cross-reactivity concerns when using ACADS antibodies?

When working with ACADS antibodies, researchers should be aware of potential cross-reactivity with other members of the acyl-CoA dehydrogenase family, which share structural similarities . These enzymes catalyze similar reactions but with different chain-length specificities.

To address cross-reactivity concerns:

• Perform comparative western blot analysis against tissues known to express varying levels of different acyl-CoA dehydrogenase family members.

• Include appropriate negative controls, such as tissues or cell lines with confirmed ACADS knockdown/knockout.

• Consider using monoclonal antibodies for higher specificity when cross-reactivity is a significant concern .

• Validate antibody specificity through orthogonal approaches like mass spectrometry to confirm identity of the detected protein .

How do post-translational modifications affect ACADS antibody recognition?

ACADS undergoes significant post-translational processing, including proteolytic cleavage when transported into mitochondria (from a 44 kDa precursor to a 41 kDa mature form) . This processing may affect antibody recognition depending on the epitope location.

Researchers should consider:

• The location of the epitope recognized by the antibody (N-terminal, C-terminal, or internal sequence).

• Potential masking of epitopes due to protein folding or complex formation.

• The impact of experimental conditions (reducing vs. non-reducing, denatured vs. native) on epitope accessibility.

• When investigating mitochondrial targeting and processing, using antibodies that recognize different regions of ACADS may provide complementary information about the processing state.

What are the optimal sample preparation methods for ACADS detection?

For optimal ACADS detection, sample preparation should consider its mitochondrial localization and properties:

For Western Blot analysis:

• Use RIPA buffer supplemented with protease inhibitors for effective extraction from mitochondria.

• Include proper reducing agents to maintain protein structure.

• Heat samples at 95°C for 5 minutes in sample buffer for denaturation.

• Load 20-50 μg of total protein per lane for detection.

For Immunohistochemistry:

• Perform heat-mediated antigen retrieval with TE buffer pH 9.0 for optimal results .

• Alternatively, citrate buffer pH 6.0 may be used for antigen retrieval .

• Use freshly prepared or properly stored fixed tissues to avoid epitope degradation.

• Include positive control tissues (kidney, heart, or liver) with known ACADS expression .

For Immunoprecipitation:

• Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate .

• Consider VeriBlot for IP Detection Reagent for improved detection specificity .

How can researchers troubleshoot inconsistent results with ACADS antibodies?

When encountering inconsistent results with ACADS antibodies, consider these troubleshooting approaches:

• Weak or no signal issues:

  • Increase antibody concentration or incubation time

  • Optimize antigen retrieval conditions (for IHC)

  • Ensure sample integrity through housekeeping protein detection

  • Verify ACADS expression in your sample type

• High background or non-specific binding:

  • Increase blocking time or blocking agent concentration

  • Decrease primary antibody concentration

  • Use more stringent washing conditions

  • Consider monoclonal alternatives if using polyclonal antibodies

• Unexpected molecular weight:

  • Verify if detecting the precursor (44 kDa) or mature form (41-42 kDa)

  • Check for potential post-translational modifications

  • Ensure reducing conditions are appropriate

• Varied results across experiments:

  • Standardize sample preparation protocols

  • Use consistent antibody lots when possible

  • Include positive controls in each experiment

How can researchers ensure reproducibility when using ACADS antibodies across studies?

Ensuring reproducibility with ACADS antibodies requires rigorous documentation and standardization:

• Antibody identification:

  • Record complete antibody information including catalog number, clone, lot number, and RRID (Research Resource Identifier)

  • Consider registering antibodies in the ABCD database for standardized identification

• Validation documentation:

  • Document validation methods used for each application

  • Include detailed imaging parameters for microscopy-based methods

  • Maintain records of positive and negative controls

• Protocol standardization:

  • Create detailed SOPs for each application

  • Include specific instrument settings and acquisition parameters

  • Document exact buffer compositions and preparation methods

• Reporting standards:

  • Follow guidelines for antibody reporting in publications

  • Include validation data in supplementary materials

  • Specify exact experimental conditions that may affect results

What are the most effective controls for validating ACADS antibody experiments?

Effective controls for ACADS antibody experiments include:

• Positive controls:

  • Tissue samples with confirmed high ACADS expression (liver, kidney, heart)

  • Cell lines with verified ACADS expression

• Negative controls:

  • ACADS knockout or knockdown samples when available

  • Isotype controls for immunohistochemistry/immunofluorescence

  • Secondary antibody-only controls

• Specificity controls:

  • Pre-absorption with immunizing peptide where available

  • Competing peptide assays to demonstrate specificity

  • Multiple antibodies targeting different epitopes of ACADS

• Technical controls:

  • Loading controls for western blot (housekeeping proteins)

  • Internal controls for IHC (tissues with known expression patterns)

  • Processing controls (samples processed identically except for antibody incubation)

How can ACADS antibodies be used to investigate mitochondrial fatty acid metabolism disorders?

ACADS antibodies provide valuable tools for investigating Short-Chain Acyl-CoA Dehydrogenase Deficiency (SCADD) and related disorders:

• Diagnostic applications:

  • Immunohistochemical assessment of ACADS protein levels in patient samples

  • Western blot analysis to detect alterations in ACADS protein size or abundance

  • Comparison of ACADS levels between affected and unaffected tissues

• Mechanistic studies:

  • Immunofluorescence co-localization with other mitochondrial markers

  • Tracking ACADS subcellular localization under metabolic stress conditions

  • Investigating post-translational modifications affecting enzyme function

• Therapeutic development:

  • Monitoring ACADS protein levels during experimental therapies

  • Assessing restoration of proper subcellular localization following interventions

  • Evaluating protein-protein interactions affecting ACADS function

• Model validation:

  • Characterizing ACADS expression in animal or cellular disease models

  • Confirming knockdown/knockout efficiency in engineered model systems

  • Validating protein-level changes predicted by transcriptomic studies

What strategies can improve detection sensitivity for low-abundance ACADS in research samples?

For detecting low-abundance ACADS, researchers can employ several strategies:

• Signal amplification methods:

  • Use tyramide signal amplification (TSA) for immunohistochemistry/immunofluorescence

  • Employ enhanced chemiluminescence (ECL) substrates with extended exposure times for western blot

  • Consider biotin-streptavidin amplification systems

• Sample enrichment techniques:

  • Isolate mitochondrial fractions to concentrate ACADS

  • Use immunoprecipitation to enrich target protein before analysis

  • Employ subcellular fractionation to reduce sample complexity

• Optimized detection systems:

  • Use highly-sensitive detection antibodies

  • Employ cooled CCD cameras for western blot imaging

  • Consider fluorescent secondary antibodies with appropriate wavelengths to minimize background

• Protocol modifications:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize blocking conditions to improve signal-to-noise ratio

  • Use smaller pore-size membranes for western blots of lower molecular weight proteins