ACADS Antibody

What is ACADS and why is it a significant target for immunodetection in metabolic research?

ACADS (Short-chain specific acyl-CoA dehydrogenase) is a key enzyme that catalyzes the first step of mitochondrial fatty acid beta-oxidation, an aerobic process that breaks down fatty acids into acetyl-CoA for energy production. ACADS specifically acts on acyl-CoAs with saturated 4 to 6 carbons long primary chains . This enzyme is particularly valuable as a research target because:

• It functions as a critical component in maintaining energy homeostasis, especially during fasting or increased energy demands

• Expression occurs in the mitochondria of various tissues, with highest levels in liver and muscle where fatty acid metabolism is most active

• Dysfunction is associated with metabolic disorders, making it relevant for studies on fatty acid oxidation disorders and mitochondrial dysfunction

For robust experimental design, researchers should consider tissue-specific expression patterns when selecting positive controls, with liver and heart tissues showing consistently high ACADS expression levels suitable for validation studies.

How should researchers select between different types of ACADS antibodies for specific applications?

Selection of appropriate ACADS antibodies should be based on both the intended application and the specific experimental requirements:

For application-specific optimization:

• Western blotting: Use 1:500-1:2000 dilution for polyclonal and 1:1000 for monoclonal antibodies

• IHC-P: Heat-mediated antigen retrieval with citrate buffer pH 6 is essential for most antibodies

• Immunofluorescence: Select antibodies explicitly validated for this application (e.g., ab110318, ABIN7007231)

When analyzing potentially contradictory results between different antibodies, consider epitope accessibility differences in your specific sample preparation method.

What validation approaches are essential before using ACADS antibodies in critical experiments?

Rigorous validation of ACADS antibodies is crucial given the finding that "most commercial antibodies fail to recognize their target proteins or bind off-target in at least some experimental applications" . A comprehensive validation strategy includes:

• Western blot characterization:

  • Confirm single band at expected molecular weight (predicted: 44 kDa, often observed: 41 kDa)

  • Test positive control samples (HeLa, HepG2, 293T cells; mouse/rat liver)

  • Compare multiple antibodies targeting different epitopes

• Specificity confirmation:

  • Use ACADS CRISPR/Cas9 knockout samples as negative controls

  • Perform gene silencing (siRNA/shRNA) to confirm signal reduction

  • Pre-absorb antibody with recombinant ACADS protein

• Application-specific validation:

  • For IHC/ICC: Confirm mitochondrial localization pattern

  • For IP: Verify target protein identity by mass spectrometry

  • For multiple applications: Validate independently for each method

• Cross-reactivity assessment:

  • Test across relevant species (human/mouse/rat) if comparative studies planned

  • Check for reactivity against other acyl-CoA dehydrogenase family members

Researchers should document validation evidence, as organizations like YCharOS have reported "a substantial fraction of antibodies performed poorly" in systematic validation studies .

How can researchers distinguish between ACADS and other acyl-CoA dehydrogenase family members in experimental systems?

Differentiating ACADS from other family members requires strategic approaches due to sequence and structural similarities:

• Epitope selection strategy: Choose antibodies targeting unique regions of ACADS. For example, antibody ab156571 uses a recombinant fusion protein containing amino acids 1-260 of human ACADS (NP_000008.1) , while CAB0945 targets a sequence corresponding to amino acids 1-260 .

• Molecular weight discrimination:

  • ACADS: Predicted at 44 kDa, typically observed at 41 kDa

  • Medium-chain acyl-CoA dehydrogenase (MCAD): ~47 kDa

  • Long-chain acyl-CoA dehydrogenase (LCAD): ~48 kDa

  • Very long-chain acyl-CoA dehydrogenase (VLCAD): ~70 kDa

• Advanced validation approaches:

  • Utilize CRISPR/Cas9 ACADS knockout cells as negative controls

  • Perform substrate-specific enzyme activity assays to confirm functional identity

  • Employ proteomic analysis to confirm antibody target identity

• Comparative expression analysis:

  • Use multiple antibodies targeting different epitopes

  • Compare with mRNA expression patterns across tissues

  • Assess tissue distribution patterns characteristic of each family member

For experimental design, researchers should include both positive controls (tissues with known ACADS expression like liver) and negative controls (knockout samples) to ensure signal specificity.

What are the critical factors affecting ACADS detection in different tissue types and experimental conditions?

Several factors significantly influence ACADS detection across diverse experimental systems:

• Tissue-specific considerations:

  • Expression variability: ACADS shows highest expression in liver, heart, and muscle tissues

  • Background interference: Endogenous biotin/peroxidase activity varies by tissue

  • Fixation sensitivity: Overfixation can mask epitopes in mitochondria-rich tissues

• Sample preparation factors:

  • Antigen retrieval requirements: Heat-mediated retrieval with citrate buffer pH 6 is critical for most ACADS antibodies in FFPE tissues

  • Extraction methods: Mitochondrial protein extraction efficiency varies by protocol

  • Post-translational modifications: Tissue-specific modifications may alter epitope accessibility

• Analysis of contradictory results:

  • Discrepancies between antibodies may reflect epitope-specific accessibility

  • Signal intensity differences between tissues may represent extraction efficiency variations

  • Consider nonspecific binding, particularly in lipid-rich tissues

• Optimization strategies:

  • For low-expressing tissues: Increase antibody concentration, extend incubation time

  • For high background: Enhance blocking (5% NFDM/TBST recommended)

  • For inconsistent results: Standardize fixation time, extraction protocol, and antibody lot

Researchers should implement a systematic approach to protocol optimization, testing multiple conditions in parallel to identify optimal parameters for their specific experimental system.

How should researchers approach ACADS antibody-based studies across different species?

Cross-species ACADS studies require careful consideration of sequence conservation and antibody validation:

• Species reactivity analysis:
  Based on the search results, antibodies show variable cross-reactivity:

  Antibody	Human	Mouse	Rat	Other Species
  ab156571	✓	✓	✓	Not specified
  CAB0945	✓	✓	✓	Not specified
  PA5-54580	✓	✓ (88% homology)	✓ (89% homology)	Not specified
  ABIN1491430	✓	-	-	Monkey (✓)

• Sequence homology considerations:

  • PA5-54580 shows 88% sequence identity to mouse ACADS and 89% to rat ACADS

  • Epitope sequence conservation should be verified before cross-species studies

• Validation requirements for comparative studies:

  • Independent validation in each species is essential

  • Species-specific positive controls must be included

  • Signal intensity variations may reflect antibody affinity differences rather than expression levels

• Experimental design for multi-species studies:

  • Use consistent sample preparation protocols across species

  • Include species-specific positive and negative controls in each experiment

  • Consider complementary techniques (qPCR, enzyme activity) to support findings

• Data interpretation challenges:

  • Signal differences may reflect antibody affinity rather than biological differences

  • Species-specific post-translational modifications may affect detection

  • Different optimal antibody concentrations may be required for each species

For resolving contradictory cross-species data, researchers should employ multiple antibodies targeting different epitopes and correlate findings with functional assays.

What are the optimal protocols for Western blot analysis of ACADS protein?

Based on published methodologies, the following optimized Western blot protocol for ACADS detection is recommended:

• Sample preparation:

  • Extract proteins from fresh/frozen tissue or cultured cells with RIPA buffer containing protease inhibitors

  • Positive controls: HeLa, HepG2, 293T cells; mouse/rat liver or heart tissue

  • Load 10-20 μg total protein per lane

• Electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels suitable for ~44 kDa proteins

  • Transfer to PVDF or nitrocellulose membrane (wet transfer recommended for mitochondrial proteins)

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST (5% NFDM/TBST)

  • Primary antibody dilutions:

    • ab156571: 1:1000

    • CAB0945: 1:500-1:2000

    • ab110318: 1 μg/mL

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

• Detection and visualization:

  • Use HRP-conjugated secondary antibodies with ECL detection

  • For low abundance samples, consider enhanced chemiluminescence substrates

  • Expected band size: Predicted 44 kDa, often observed at 41 kDa

• Troubleshooting recommendations:

  • For multiple bands: Increase blocking stringency, optimize antibody dilution

  • For high background: Extend washing steps, reduce antibody concentration

  • For weak signal: Increase protein loading, extend exposure time, or try signal enhancement systems

For quantitative analysis, include both technical replicates and appropriate loading controls, preferably other mitochondrial proteins to normalize for mitochondrial content.

What are the key considerations for immunohistochemical detection of ACADS in tissue sections?

Optimized immunohistochemistry protocol for ACADS detection based on published methods:

• Tissue preparation:

  • Fixation: 10% neutral buffered formalin fixation (10-24 hours)

  • Processing: Standard paraffin embedding

  • Sectioning: 4-6 μm thickness on positively charged slides

• Antigen retrieval optimization:

  • Heat-mediated antigen retrieval with citrate buffer pH 6 is consistently recommended across antibodies

  • Alternative: Tris-EDTA buffer pH 9.0 for certain antibodies (ab156571)

  • Critical parameter: Maintain optimal temperature (95-98°C) for 15-20 minutes

• Staining protocol:

  • Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes

  • Protein blocking: Serum-free protein block, 30 minutes

  • Primary antibody dilutions:

    • ab156571: 1:50-1:100

    • CAB0945: 1:50-1:100

  • Incubation time: 60 minutes at room temperature or overnight at 4°C

  • Detection system: HRP-polymer detection systems recommended

  • Counterstain: Hematoxylin for nuclear visualization

• Controls and validation:

  • Positive tissue controls: Liver, heart, and colon show consistent positivity

  • Negative controls: Primary antibody omission and ACADS-negative tissues

  • Expected pattern: Mitochondrial (cytoplasmic, granular) staining pattern

• Technical considerations:

  • Tissue thickness affects staining intensity and background

  • Freshly cut sections provide superior results compared to stored slides

  • Automated platforms require protocol optimization specific to each system

For quantitative analysis of immunohistochemical staining, implement digital pathology approaches with defined scoring systems and analyze multiple fields per sample to account for heterogeneity.

How should researchers approach troubleshooting when ACADS antibodies produce unexpected results?

Systematic troubleshooting framework for addressing common challenges with ACADS antibodies:

• No signal or weak signal issues:

  Problem	Probable Causes	Methodological Solutions
  No Western blot signal	Inefficient extraction, epitope destruction	Use mitochondria-specific extraction buffers, reduce sample heating
  No IHC/IF signal	Inadequate antigen retrieval, overfixation	Optimize antigen retrieval conditions, test multiple fixation times
  Weak signal across applications	Low expression, antibody degradation	Use amplification systems, test fresh antibody aliquots

• Non-specific or high background issues:

  Problem	Probable Causes	Methodological Solutions
  Multiple WB bands	Cross-reactivity, protein degradation	Increase blocking time/stringency, add protease inhibitors during extraction
  Diffuse IHC staining	Overfixation, excessive antibody	Optimize fixation protocol, titrate antibody concentration
  High membrane background	Insufficient blocking, washing issues	Use 5% NFDM/TBST , add 0.1% Tween-20 to wash buffers

• Contradictory results analysis:

  Contradiction Type	Analytical Approach	Resolution Strategy
  Different antibodies show varied results	Compare epitope locations, clonality	Test multiple antibodies targeting different regions
  Signal in WB but not IHC	Epitope accessibility differences	Try alternative fixation/retrieval methods
  Unexpected tissue distribution	Potential cross-reactivity	Validate with knockout controls, compare with mRNA expression

• Advanced validation for problem resolution:

  • Implement ACADS CRISPR/Cas9 knockout controls for definitive specificity testing

  • Consider alternative ACADS detection methods (enzyme activity assays)

  • Perform epitope mapping to identify potential cross-reactive regions

For resolving persistent issues, the search results emphasize that "there may not be one solution. But there may be many" , suggesting combinatorial approaches may be necessary.

What methods can researchers use to quantify ACADS protein expression levels?

Robust quantification of ACADS requires appropriate methodological approaches for different experimental systems:

• Western blot quantification methodologies:

  • Establish standard curves using recombinant ACADS protein

  • Use mitochondrial housekeeping proteins (VDAC, COX4) as loading controls

  • Employ digital image analysis with background subtraction

  • Ensure detection within linear range of signal

  • Report relative expression normalized to mitochondrial markers

• Immunohistochemistry quantification approaches:

  • Implement standardized scoring systems (H-score, Allred score)

  • Use digital pathology software for unbiased quantification

  • Analyze multiple fields (minimum 5-10) per sample

  • Consider automated image analysis algorithms

  • Report both intensity and distribution parameters

• ELISA-based absolute quantification:

  • Commercial ACADS ELISA kits utilize validated antibodies like CAB0945

  • Generate standard curves with recombinant ACADS protein

  • Process all experimental samples simultaneously

  • Account for matrix effects with appropriate dilutions

  • Apply four-parameter logistic regression for curve fitting

• Flow cytometry for cellular analysis:

  • Require permeabilization protocols optimized for mitochondrial targets

  • Use median fluorescence intensity (MFI) as quantitative measure

  • Include fluorescence calibration beads for standardization

  • Establish negative thresholds with isotype controls

• Statistical considerations:

  • Perform multiple technical and biological replicates

  • Apply appropriate statistical tests for experimental design

  • Consider nonparametric methods for IHC scoring data

  • Report variability measures (standard deviation, confidence intervals)

When comparing ACADS levels across different experimental models or tissues, normalize to mitochondrial content rather than total protein to account for variations in mitochondrial abundance.

How can researchers optimize co-localization studies involving ACADS antibodies?

Strategic approaches for designing and analyzing ACADS co-localization experiments:

For analyzing contradictory co-localization data, implement both visual assessment and multiple quantitative metrics, as different coefficients may reveal distinct aspects of spatial relationships between proteins.