ACADVL Antibody

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

ACADVL (also known as VLCAD) is a critical enzyme that catalyzes the first step of mitochondrial fatty acid beta-oxidation, an aerobic process that breaks down fatty acids into acetyl-CoA to enable energy production from fats. This enzyme specifically removes hydrogen from C-2 and C-3 positions of straight-chain fatty acyl-CoA thioesters, creating trans-2-enoyl-CoA products . ACADVL is distinctive among acyl-CoA dehydrogenases for its specificity toward acyl-CoAs with saturated 12 to 24 carbon long primary chains . This enzyme's function is crucial to understanding mitochondrial metabolism disorders, particularly in tissues with high energy demands such as cardiac and skeletal muscle.

What criteria should researchers use when selecting an ACADVL antibody?

When selecting an ACADVL antibody, researchers should consider:

• Antibody type and specificity: Choose between monoclonal (e.g., rabbit recombinant monoclonal EPR15107(B) or mouse monoclonal H-7 ) and polyclonal options (e.g., rabbit polyclonal 14527-1-AP ) based on experimental needs. Monoclonals offer higher specificity while polyclonals provide broader epitope recognition.

• Validated applications: Ensure the antibody has been validated for your intended application:

  • Western blot (1:1000-1:4000 dilution)

  • Immunohistochemistry (1:100-1:400 dilution)

  • Immunoprecipitation

  • Flow cytometry

  • Co-immunoprecipitation

• Species reactivity: Confirm reactivity with your experimental model (human, mouse, rat) .

• Validation data: Review knockout validation data where available. For example, ab188872 has demonstrated specificity through testing with ACADVL knockout HEK293T cell lines .

What are the optimal conditions for using ACADVL antibodies in Western blot applications?

For Western blotting with ACADVL antibodies:

• Sample preparation: Use fresh tissue lysates from liver, muscle, or heart, or cell lines like HEK293T, HeLa, or HepG2 .

• Protein loading: Load 20μg of total protein per lane for optimal signal detection .

• Recommended dilutions:

  • Rabbit monoclonal antibodies (e.g., EPR15107(B)): 1:1000-1:10000

  • Rabbit polyclonal antibodies: 1:1000-1:4000

• Expected molecular weight: 70 kDa (predicted), with observed bands typically between 66-73 kDa .

• Controls: Include positive controls (e.g., liver tissue) and negative controls (ACADVL knockout cell lysates where available) .

• Secondary antibody options: For dual detection systems, use appropriate secondary antibodies such as:

  • Goat anti-Rabbit IgG H&L (IRDye® 800CW) at 1:10000-1:20000 dilution

  • HRP-conjugated secondaries at 1:1000 dilution

How should researchers optimize immunohistochemistry protocols with ACADVL antibodies?

For optimal IHC results with ACADVL antibodies:

• Antigen retrieval: Perform heat-mediated antigen retrieval using:

  • Primary option: Tris/EDTA buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

• Antibody dilution: Use 1:100-1:500 dilution depending on the specific antibody .

• Detection systems:

  • For rabbit antibodies: Pre-diluted HRP Polymer for Rabbit IgG followed by hematoxylin counterstaining

  • For mouse antibodies: Appropriate anti-mouse detection systems

• Tissue selection: ACADVL shows strong expression in metabolically active tissues:

  • Human kidney tissue

  • Human liver tissue

  • Colon tissue

• Controls: Include tissues known to express ACADVL (liver, heart) and consider negative controls using isotype control antibodies.

How can ACADVL antibodies be used in co-immunoprecipitation studies to investigate protein interactions?

For co-immunoprecipitation (Co-IP) studies with ACADVL antibodies:

• Antibody selection: Choose antibodies specifically validated for IP applications, such as ab188872 (used at 1:50 dilution for immunoprecipitation) or polyclonal antibody 14527-1-AP (validated for CoIP) .

• Cell/tissue preparation:

  • Use freshly prepared lysates from A431 cells or other relevant cell lines

  • Employ gentle lysis buffers containing protease inhibitors to preserve protein-protein interactions

  • Pre-clear lysates to reduce non-specific binding

• Immunoprecipitation protocol:

  • Incubate antibody with lysate (typically 1-5 μg antibody per mg of total protein)

  • Capture complexes using Protein A/G beads

  • Wash extensively to remove non-specific interactions

  • Elute and analyze by Western blot using antibodies against potential interaction partners

• Controls: Include:

  • IgG control (same species as the primary antibody)

  • Input sample (pre-immunoprecipitation lysate)

  • Reverse Co-IP validation where possible

How can researchers validate ACADVL antibody specificity in experimental models?

Validation strategies for ACADVL antibodies include:

• Knockout validation:

  • Test antibodies against ACADVL knockout cell lines (e.g., Human ACADVL knockout HEK-293T cell line ab266484)

  • This provides definitive evidence of specificity when signal is lost in knockout samples

• Genetic models:

  • CRISPR/Cas9-generated models with ACADVL variants can be used to confirm antibody specificity and study variant effects

  • These models are particularly valuable when studying variants of uncertain significance

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide

  • Loss of signal confirms specificity for the target epitope

• Multiple antibody validation:

  • Use antibodies from different sources recognizing different epitopes

  • Consistent results across different antibodies provide confidence in specificity

• Multiple detection methods:

  • Confirm findings using both IHC and WB techniques

  • Correlate with mRNA expression data where possible

How can ACADVL antibodies contribute to studies of VLCAD deficiency and related metabolic disorders?

ACADVL antibodies are invaluable tools for studying VLCAD deficiency:

• Protein expression analysis:

  • Quantify ACADVL protein levels in patient samples versus controls

  • Assess expression in different tissues to understand disease pathophysiology

• Variant characterization:

  • With >300 variants of uncertain significance (VUSs) in the ACADVL gene requiring characterization , antibodies can help determine how these variants affect protein expression and stability

  • Combined with CRISPR/Cas9 gene editing, antibodies can help establish genotype-phenotype correlations

• Diagnostic research:

  • Complement genetic testing approaches, which detect variants within coding regions and intron-exon boundaries

  • Study protein expression patterns in tissues from affected individuals

• Therapeutic development:

  • Monitor protein expression changes in response to experimental therapies

  • Evaluate restoration of enzyme function in treatment models

• Model systems:

  • Validate animal and cellular models of VLCAD deficiency

  • Study tissue-specific effects of ACADVL mutations

What are the best practices for using ACADVL antibodies in flow cytometry applications?

For flow cytometry applications with ACADVL antibodies:

• Cell preparation:

  • Fix cells with paraformaldehyde (2%) for intracellular staining

  • Permeabilize cell membranes to allow antibody access to this mitochondrial protein

• Antibody dilution:

  • Use ab188872 at 1:160 dilution for intracellular flow cytometry

  • Other antibodies should be titrated to determine optimal concentration

• Controls:

  • Include isotype controls (e.g., rabbit IgG negative control)

  • Use cells with known ACADVL expression levels as positive controls

• Secondary antibody selection:

  • For rabbit primary antibodies, use anti-rabbit IgG (FITC) at approximately 1:150 dilution

  • Ensure secondary antibodies are compatible with flow cytometry applications

• Gating strategy:

  • Include mitochondrial markers for co-localization studies

  • Use appropriate controls to set gates for positive and negative populations

How can droplet digital PCR (ddPCR) complement antibody-based detection of ACADVL in research studies?

While antibody-based detection provides insights into protein expression and localization, ddPCR offers complementary genomic information:

• Copy number analysis:

  • ddPCR can be used to determine genomic copy number of ACADVL exons and introns

  • This approach helps identify large deletions or duplications that may not be detected by standard sequencing

• Integration with antibody studies:

  • Correlate genomic copy number variations with protein expression levels detected by antibodies

  • Multiplex ddPCR assays can target specific ACADVL regions (e.g., intron 11 and exons 10, 15, or 20)

• Protocol considerations:

  • Use restriction enzyme digestion (e.g., EcoRI-HF) of genomic DNA before ddPCR

  • Utilize appropriate probes (e.g., FAM-labeled for ACADVL regions and HEX-labeled for reference loci like RPP30)

  • Calculate genomic copy number by comparing target concentration to reference loci

What considerations are important when using ACADVL antibodies in multi-omics research approaches?

When integrating ACADVL antibody-based studies into multi-omics approaches:

• Proteomics integration:

  • Use antibodies for immunoprecipitation followed by mass spectrometry to identify interaction partners

  • Compare protein expression levels from antibody-based techniques with shotgun proteomics data

• Genomics correlation:

  • Connect antibody-detected protein expression patterns with genomic variants identified through sequencing

  • Consider how variants (pathogenic, likely pathogenic, or VUS) affect protein expression

• Transcriptomics relationship:

  • Compare protein levels detected by antibodies with mRNA expression levels

  • Investigate post-transcriptional regulation mechanisms when discrepancies are observed

• Metabolomics connections:

  • Correlate ACADVL protein levels with metabolite profiles, particularly long-chain fatty acids and acylcarnitines

  • Investigate how protein expression variations impact metabolic pathways

• Tissue-specific considerations:

  • Account for tissue-specific expression patterns when designing multi-omics studies

  • Select appropriate antibody dilutions based on expected expression levels in different tissues