ACAD8 Antibody

What is ACAD8 and why is it important in metabolic research?

ACAD8, also known as isobutyryl-CoA dehydrogenase (IBD), is a nuclear-encoded mitochondrial enzyme belonging to the acyl-CoA dehydrogenase family (EC 1.3.99). This enzyme plays a critical role in the metabolism of fatty acids and branched chain amino acids, specifically catalyzing the conversion of isobutyryl-CoA to methacrylyl-CoA in the valine catabolism pathway . ACAD8's importance in metabolism research stems from its central position in essential catabolic pathways and its association with metabolic disorders. Deficiencies in ACAD8 function have been linked to elevated C4-carnitine levels in newborns, a biomarker for potential metabolic disorders . The protein exists in three isoforms with molecular weights of approximately 45 kDa, 31 kDa, and 38 kDa, providing researchers with multiple targets for investigation across various experimental contexts . Understanding ACAD8 function contributes significantly to our knowledge of mitochondrial metabolism and inborn errors of metabolism.

What applications are ACAD8 antibodies validated for?

ACAD8 antibodies have been validated for multiple research applications, allowing for comprehensive protein analysis across various experimental platforms. Based on available data, these antibodies demonstrate utility in the following applications:

These applications enable researchers to investigate ACAD8 expression patterns, subcellular localization, protein-protein interactions, and alterations in various experimental conditions or disease states . It is recommended that researchers titrate antibody concentrations within the suggested ranges to determine optimal conditions for their specific experimental system, as sensitivity can vary depending on sample type and preparation method .

How should researchers select the appropriate ACAD8 antibody for their experiments?

Selection of an appropriate ACAD8 antibody should be based on several critical factors that impact experimental outcomes. Researchers should consider:

• Species reactivity: Available ACAD8 antibodies demonstrate reactivity with human, mouse, and rat samples . Verify that your chosen antibody recognizes ACAD8 in your species of interest.

• Epitope specificity: Different antibodies target distinct regions of the ACAD8 protein. For example:

  • AA 201-415 (ABIN7157099)

  • AA 1-415 (multiple vendors)

  • AA 118-145 (available options)

  • AA 77-126 (available options)

• Clonality: Both monoclonal and polyclonal options are available:

  • Polyclonal antibodies: Higher sensitivity, recognize multiple epitopes

  • Monoclonal antibodies: Greater specificity, more consistent across batches

• Application compatibility: Ensure the antibody is validated for your desired application (WB, IHC, ELISA, IP) .

• Form and conjugation: Available in unconjugated form or conjugated with:

  • FITC (for direct immunofluorescence)

  • HRP (for direct enzymatic detection)

  • Biotin (for streptavidin-based detection systems)

• Validation data: Review validation data showing the expected molecular weight detection (45 kDa for the main isoform) and sample-specific reactivity.

What are the optimal sample preparation protocols for Western blotting with ACAD8 antibodies?

For optimal results in Western blotting experiments with ACAD8 antibodies, researchers should follow these methodological guidelines:

• Sample preparation:

  • Prepare lysates from validated positive tissues (e.g., mouse pancreas tissue, mouse lung tissue)

  • Use appropriate lysis buffers containing protease inhibitors to prevent degradation

  • For mitochondrial proteins like ACAD8, consider specialized mitochondrial extraction protocols

• Protein quantification and loading:

  • Standardize protein concentration using reliable methods (BCA, Bradford)

  • Load 20-50 μg total protein per lane, adjusting based on expression level

  • Include positive control samples where ACAD8 expression is established

• Gel electrophoresis conditions:

  • Use 10-12% polyacrylamide gels for optimal resolution around 45 kDa

  • Include molecular weight markers covering the 30-50 kDa range to identify all isoforms

• Transfer parameters:

  • Optimize transfer conditions for proteins in the 45 kDa range

  • Consider semi-dry or wet transfer methods based on your equipment

• Antibody incubation:

  • Block membranes thoroughly (typically 5% non-fat milk or BSA in TBST)

  • Dilute primary antibody in recommended range (1:500-1:2000)

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

  • Use appropriate HRP-conjugated secondary antibody at manufacturer's recommended dilution

• Detection system:

  • Use enhanced chemiluminescence (ECL) substrates appropriate for your expected protein abundance

  • Optimize exposure times to prevent signal saturation

• Controls:

  • Include negative controls (non-expressing tissues or knockdown samples)

  • Consider using recombinant ACAD8 protein as a positive control

How can researchers validate the specificity of ACAD8 antibodies in their experimental systems?

Antibody validation is critical for ensuring experimental reproducibility and accuracy. For ACAD8 antibodies, researchers should implement a multi-faceted validation strategy:

• Genetic modification approaches:

  • CRISPR/Cas9 knockout: Generate ACAD8 knockout cell lines to confirm antibody specificity

  • siRNA/shRNA knockdown: Perform transient knockdown to verify signal reduction correlates with reduced expression

  • Overexpression: Transfect cells with ACAD8 expression constructs to confirm increased signal

• Immunological validation:

  • Peptide competition assays: Pre-incubate antibody with immunogen peptide to block specific binding

  • Multiple antibody validation: Compare results using antibodies targeting different ACAD8 epitopes

  • Isotype controls: Use matched isotype control antibodies to assess non-specific binding

• Analytical validation:

  • Mass spectrometry: Perform immunoprecipitation followed by mass spectrometry analysis

  • Western blot: Confirm detection at the expected molecular weight (45 kDa for main isoform)

  • Cross-reactivity assessment: Test against related ACAD family members to confirm specificity

• Tissue expression validation:

  • Compare detection patterns with established ACAD8 expression profiles

  • Verify positive signal in known ACAD8-expressing tissues (e.g., mouse pancreas, mouse lung)

  • Assess subcellular localization matches expected mitochondrial pattern

• Documentation:

  • Record comprehensive validation data including antibody catalog number (e.g., 16742-1-AP, ABIN7157099)

  • Document RRID (Research Resource Identifier) when available (e.g., AB_2219559)

  • Maintain detailed protocols for reproducibility

What are the considerations for optimizing immunohistochemistry protocols for ACAD8 detection?

Immunohistochemical detection of ACAD8 requires careful optimization of multiple parameters:

• Tissue processing considerations:

  • Fixation: 4% paraformaldehyde or 10% neutral buffered formalin (10-24 hours at 4°C)

  • Processing: Standard dehydration and paraffin embedding or freezing for cryosectioning

  • Section thickness: 4-6 μm for optimal antibody penetration and signal resolution

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER): Test multiple buffers

    • Citrate buffer (pH 6.0)

    • EDTA buffer (pH 8.0)

    • Tris-EDTA buffer (pH 9.0)

  • Enzymatic retrieval: Consider proteinase K or trypsin digestion if HIER is insufficient

  • Optimization: Adjust temperature, pressure, and duration based on tissue type

• Blocking and antibody incubation:

  • Blocking: 5-10% normal serum (from secondary antibody species) with 0.1-0.3% Triton X-100

  • Primary antibody: Dilute according to validation data, typically overnight at 4°C

  • Secondary detection: HRP-polymer systems or fluorescently-labeled secondary antibodies

  • Counterstaining: Hematoxylin for brightfield or DAPI/Hoechst for fluorescence

• Signal detection optimization:

  • Chromogenic detection: DAB development time optimization (typically 2-10 minutes)

  • Fluorescent detection: Select appropriate filters to minimize tissue autofluorescence

  • Amplification systems: Consider tyramide signal amplification for low abundance targets

• Controls and validation:

  • Positive control tissues: Mouse pancreas and lung tissue

  • Negative controls: Primary antibody omission and isotype controls

  • Subcellular localization: Confirm mitochondrial pattern using co-staining with mitochondrial markers

• Quantification considerations:

  • Standardized acquisition settings across experimental groups

  • Digital image analysis with appropriate background correction

  • Normalization to housekeeping proteins or cell counts

How do different isoforms of ACAD8 affect antibody selection and experimental design?

The presence of multiple ACAD8 isoforms (45 kDa, 31 kDa, and 38 kDa) presents both challenges and opportunities for experimental design:

• Isoform-specific detection strategies:

• Experimental considerations for isoform analysis:

  • Gel percentage: Use 10-15% gradient gels for optimal separation of all isoforms

  • Running conditions: Extend separation time for closely migrating isoforms

  • Molecular weight markers: Include precise markers in the 30-50 kDa range

  • Loading controls: Select controls that don't overlap with ACAD8 isoforms

• Tissue and cell type considerations:

  • Document tissue-specific isoform expression patterns

  • Consider developmental regulation of isoform expression

  • Evaluate species-specific isoform differences when using cross-reactive antibodies

• Functional studies:

  • Design isoform-specific knockdown/knockout strategies

  • Consider isoform-specific primer pairs for qPCR correlation

  • Investigate potential differential functions of each isoform

• Pathological significance:

  • Analyze isoform ratio changes in disease states

  • Determine if isoform-specific alterations occur in metabolic disorders

  • Consider isoform-specific interactions with other proteins

What are effective troubleshooting strategies for weak or non-specific ACAD8 antibody signals?

When encountering signal problems with ACAD8 antibodies, researchers should implement a systematic troubleshooting approach:

• Weak or absent specific signal:

• High background or non-specific binding:

• Unexpected banding patterns:

  • Verify expected molecular weight (45 kDa for main isoform)

  • Consider post-translational modifications if bands appear higher than expected

  • Evaluate for degradation products if multiple lower bands appear

  • Test antibody specificity with peptide competition assays

• Optimization strategies:

  • Use positive control tissues (mouse pancreas, mouse lung)

  • Compare multiple antibodies targeting different epitopes

  • Consider antibody purification method (affinity purification may reduce background)

  • Validate storage conditions (proper aliquoting and storage at -20°C)

How can researchers effectively use ACAD8 antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence with ACAD8 antibodies allows simultaneous detection of multiple targets, enhancing experimental insights:

• Antibody selection for multiplexing:

  • Choose antibodies from different host species to prevent cross-reactivity

  • Consider directly conjugated antibodies (FITC, HRP, biotin conjugates are available)

  • Select antibodies validated for immunofluorescence applications

• Spectral considerations:

  • Plan fluorophore combinations to minimize spectral overlap

  • For ACAD8-FITC conjugates, pair with red/far-red fluorophores for other targets

  • Include single-color controls to assess bleed-through

• Mitochondrial co-localization strategies:

  • Pair ACAD8 with established mitochondrial markers:

    • TOMM20 (outer membrane)

    • COX IV (inner membrane)

    • MnSOD (matrix)

  • Use super-resolution microscopy for precise co-localization analysis

• Sequential staining protocols:

  • For same-species antibodies, implement sequential detection with intermediate blocking

  • Consider tyramide signal amplification to allow antibody stripping between rounds

  • Use directly conjugated primary antibodies to eliminate secondary antibody cross-reactivity

• Optimization for specific tissue types:

  • Adjust fixation protocols based on tissue preservation requirements

  • Implement tissue-specific autofluorescence reduction methods

  • Optimize antigen retrieval for each target protein

• Quantitative analysis approaches:

  • Standardize image acquisition parameters across all experimental groups

  • Implement automated quantification algorithms for unbiased analysis

  • Consider co-localization coefficients for interaction studies

What considerations are important when using ACAD8 antibodies for studying metabolic disorders?

ACAD8 (isobutyryl-CoA dehydrogenase) deficiency represents an important metabolic disorder that can be investigated using ACAD8 antibodies:

• Clinical sample considerations:

  • Coordinate with clinical laboratories identifying elevated C4-carnitine in newborns

  • Establish appropriate control samples (age-matched, tissue-matched)

  • Consider ethical approvals and consent requirements for patient samples

• Genotype-phenotype correlation studies:

  • Select antibodies targeting epitopes preserved in common ACAD8 mutations

  • Consider how specific mutations might affect antibody binding sites

  • Develop methods to distinguish wild-type from mutant protein expression

• Functional analysis approaches:

  • Complement protein detection with enzyme activity assays

  • Correlate protein levels with metabolite profiles (C4-carnitine levels)

  • Develop cell-based assays to test mutation effects on protein stability

• Model system selection:

  • Choose appropriate models (patient-derived cells, animal models)

  • Verify antibody cross-reactivity with model species (human, mouse, rat reactivity available)

  • Consider tissue-specific expression patterns when selecting experimental systems

• Therapeutic monitoring applications:

  • Establish baseline expression levels in patient samples

  • Develop standardized protocols for longitudinal assessment

  • Consider how potential treatments might affect antibody epitope accessibility

How does sample preparation affect ACAD8 detection in different experimental contexts?

Sample preparation significantly impacts ACAD8 detection across different experimental platforms:

• Western blot sample preparation:

  • Lysis buffer considerations:

    • RIPA buffer: Good for general protein extraction

    • NP-40 buffer: Milder detergent for preserving protein-protein interactions

    • Specialized mitochondrial extraction buffers for enriched preparations

  • Critical additives:

    • Protease inhibitor cocktails (prevent degradation)

    • Phosphatase inhibitors (preserve phosphorylation states)

    • Reducing agents (maintain protein in reduced state)

• Immunohistochemistry/immunofluorescence preparation:

  • Fixation optimization:

    • Crosslinking fixatives (paraformaldehyde, formalin): Preserve structure but may mask epitopes

    • Precipitating fixatives (methanol, acetone): Better epitope preservation but poorer morphology

    • Fixation duration: Typically 24-48 hours for tissues, 10-15 minutes for cells

  • Processing considerations:

    • Paraffin embedding: Superior morphology but requires deparaffinization

    • Frozen sections: Better epitope preservation but challenging morphology

    • Vibratome sections: No fixative penetration issues but technically demanding

• Immunoprecipitation considerations:

  • Lysis conditions:

    • Non-denaturing conditions to preserve native protein structure

    • Mild detergents (0.5-1% NP-40 or Triton X-100)

    • Physiological salt concentrations to maintain interactions

  • Pre-clearing strategies:

    • Pre-clear lysates with protein A/G beads to reduce non-specific binding

    • Use isotype control antibodies for negative controls

• ELISA sample preparation:

  • Standardize protein extraction methods across all samples

  • Consider matrix effects when preparing tissue or cell lysates

  • Include appropriate dilution series to ensure measurements within linear range

What are the quantitative approaches for measuring ACAD8 expression levels?

Quantitative assessment of ACAD8 requires methodological rigor and appropriate controls:

• Western blot quantification:

• Immunohistochemistry quantification:

  • Digital image analysis parameters:

    • Define positive staining thresholds objectively

    • Measure staining intensity, area, and distribution

    • Normalize to tissue area or cell count

  • Controls for standardization:

    • Include reference samples in each batch

    • Use automated staining platforms when possible

    • Maintain consistent acquisition settings

• ELISA-based quantification:

  • Standard curve generation:

    • Use purified recombinant ACAD8 protein

    • Include standards on each plate

    • Ensure measurements fall within linear range

  • Sample preparation standardization:

    • Normalize to total protein concentration

    • Process all samples simultaneously

    • Include quality control samples

• Flow cytometry approaches:

  • Signal calibration:

    • Use calibration beads for standardization

    • Include unstained and single-stained controls

    • Compensate for spectral overlap in multiplex assays

  • Analysis parameters:

    • Gate on relevant cell populations

    • Report median fluorescence intensity

    • Use consistent analysis templates