ACAD11 Antibody

What is ACAD11 and what is its primary function?

ACAD11 is a member of the acyl-CoA dehydrogenase family involved in fatty acid metabolism. It plays a crucial role in energy production within cells by participating in the β-oxidation of fatty acids. Unlike other ACADs that primarily function in energy-generating tissues (muscle, heart), ACAD11 is highly expressed in human brain tissue, suggesting it may serve specialized functions beyond energy metabolism . ACAD11 specifically utilizes substrates with primary carbon chain lengths between 20 and 26, with optimal activity towards C22CoA. This substrate specificity suggests it may be involved in controlling the composition of specific fatty acids in the central nervous system or catabolizing functional intermediates of aromatic amino acids .

What are the key structural features of ACAD11?

ACAD11 has a complex gene structure with multiple predicted exons that code for several protein domains:

• A conserved ACAD catalytic domain critical for dehydrogenase activity

• A predicted aminoglycoside phosphotransferase (APH) domain (which could also be a homoserine kinase type II domain)

• A peroxisome targeting signal 1 (PTS1) at its carboxy terminus

• Shares 46% identity with ACAD10, another recently characterized ACAD

This domain structure is evolutionarily conserved, as similar arrangements are found in non-mammalian orthologues, including C. elegans .

Which tissues express ACAD11 and at what levels?

ACAD11 shows a distinct tissue expression pattern:

• Highest expression in adult brain (1.3 times the expression of housekeeping gene GUSB)

• Significant expression in adult liver, heart, and kidney

• Generally higher expression levels than ACAD10 in liver and adult brain

• Expression detected through real-time PCR using probes targeting common sequences in most identified transcripts

What applications are ACAD11 antibodies validated for?

Commercial ACAD11 antibodies are validated for multiple research applications:

• Western blotting (WB) - typically at 1:500-1:2000 dilution

• Enzyme-linked immunosorbent assay (ELISA)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF)

• Immunocytochemistry (ICC)

The specific applications vary by antibody product, with some antibodies being validated for multiple applications while others have more limited validation.

How should researchers select the appropriate ACAD11 antibody epitope for their specific application?

The choice of epitope is critical when designing experiments with ACAD11 antibodies:

When studying potential alternative splice variants of ACAD11, researchers should select antibodies targeting epitopes present in all known isoforms or choose multiple antibodies targeting different regions to detect potential differences in expression patterns .

What methodological approaches are recommended for studying ACAD11 expression in brain tissues?

Based on ACAD11's high expression in brain tissue, several methodological approaches are recommended:

• RT-PCR with specific primer design: Use primer pairs targeting unique exon junctions to measure expression levels of alternative transcripts. For ACAD11, primers p11/12 have been used successfully to target sequences common to most identified transcripts .

• Western blotting of subcellular fractions: Separate mitochondrial membrane and matrix protein fractions before immunoblotting. Previous studies used approximately 100 μg of total protein from each tissue lysate separated on 12% SDS polyacrylamide gels .

• Immunohistochemistry: For studying ACAD11 distribution in brain regions:

  • Use formalin-fixed, paraffin-embedded sections of normal cerebellum

  • Cut 5 μm sections at regular intervals

  • Perform staining using the avidin-biotinylated peroxidase complex (ABC) method

  • Use antibody dilutions of 1:200 to 1:300

  • Counterstain with hematoxylin

• Immunofluorescence co-localization: To confirm mitochondrial localization, co-stain with mitochondrial markers such as ATPase antibodies .

How can researchers distinguish between ACAD11 and other ACAD family members to ensure antibody specificity?

Ensuring antibody specificity is crucial when studying ACAD11, given its 46% homology with ACAD10 and similarity to other ACAD family members:

• Use peptide-specific antibodies: Target unique peptide sequences. For example, previous research used antibodies raised against the synthetic peptide RKGQEVLIKVKHFMK, which is unique to human ACAD11 at the predicted surface of its ACAD homologous domain .

• Validate with recombinant proteins: Express recombinant ACAD10 and ACAD11 proteins in E. coli expression systems (such as pET-21a(+) vector) to test antibody cross-reactivity .

• Perform competition assays: Pre-incubate the antibody with excess purified ACAD11 antigen before immunodetection to confirm binding specificity.

• Include appropriate controls: Use tissues with known differential expression of ACAD family members (e.g., brain tissue for ACAD11 vs. muscle tissue for MCAD) to confirm specificity in a biological context .

What are the recommended protocols for co-immunoprecipitation studies involving ACAD11?

For co-immunoprecipitation studies investigating ACAD11 protein interactions:

• Cell/tissue preparation:

  • For brain tissue: Homogenize in non-denaturing lysis buffer containing 1% n-Dodecyl β-D-maltoside detergent (similar to protocols used for ACAD expression studies)

  • For cultured cells: Use neuroblastoma SK-N-SH cells as they express ACAD11

• Antibody binding:

  • Use 2-5 μg of anti-ACAD11 antibody per 500 μg of protein lysate

  • Incubate overnight at 4°C with gentle rotation

• Precipitation:

  • Add Protein A/G beads and incubate for 2-4 hours

  • Wash 4-5 times with cold lysis buffer containing reduced detergent (0.1%)

  • Elute with 2X SDS sample buffer at 95°C for 5 minutes

• Western blot analysis:

  • Use 12% SDS-PAGE to separate proteins

  • Transfer to nitrocellulose membrane

  • Probe with antibodies against suspected interaction partners

How can researchers investigate the role of ACAD11 in fatty acid metabolism using antibody-based approaches?

To study ACAD11's role in fatty acid metabolism, particularly in brain tissue:

• Subcellular localization:

  • Perform cell fractionation to separate mitochondria and peroxisomes (noting ACAD11's PTS1 domain)

  • Use digitonin (25 μg/ml) for selective permeabilization to distinguish organelle localization

  • Immunostain with ACAD11 antibodies and organelle markers

• Expression correlation:

  • Use real-time PCR with normalization to housekeeping genes (GUSB or 18S rRNA) using the ΔΔC_T method to correlate transcript and protein levels

  • Perform Western blot analysis of ACAD11 alongside other fatty acid metabolism enzymes

• Functional studies:

  • Perform enzymatic assays using immunoprecipitated ACAD11 with various substrate chain lengths (C20-C26)

  • Monitor activity with the electron transfer flavoprotein fluorescence reduction assay

• ACAD profiling:

  • Use antibodies against multiple ACADs (ACAD9, ACAD10, ACAD11, MCAD, VLCAD) to create activity profiles in different brain regions and correlate with specific fatty acid substrates

What are the common challenges when using ACAD11 antibodies and how can they be addressed?

Several challenges may arise when working with ACAD11 antibodies:

• Multiple isoforms detection: ACAD11 may have alternative splice variants that could be missed by certain antibodies. Solution: Use multiple antibodies targeting different regions or design experiments to specifically detect alternative transcripts using real-time PCR with primer pairs targeted to unique exon junctions .

• Cross-reactivity with ACAD10: Due to 46% sequence identity between ACAD10 and ACAD11, cross-reactivity may occur. Solution: Use peptide-specific antibodies targeting unique regions or validate specificity using recombinant proteins and knockout/knockdown controls .

• Low signal in certain applications: Solution: Optimize antibody concentration (typically 1:500-1:2000 for WB), incubation conditions, and detection methods. For tissues with lower expression, consider signal amplification techniques .

• Background in brain tissue: Brain tissue can yield high background staining. Solution: Increase blocking duration, optimize antibody dilution, and use antigen retrieval techniques appropriate for fixed brain tissue.

How should researchers validate ACAD11 antibody specificity in their experimental systems?

Thorough validation is essential for obtaining reliable results:

• Positive controls: Use tissues with known high expression of ACAD11 (brain, liver) or cell lines such as HepG2, 293T, or Raji cells that have been validated as positive samples .

• Peptide competition: Pre-incubate the antibody with excess immunizing peptide to confirm signal specificity.

• Molecular weight verification: Confirm that the detected band matches the expected molecular weight of ACAD11 (approximately 87 kDa for the full-length protein).

• RNA interference: Perform siRNA or shRNA knockdown of ACAD11 and demonstrate corresponding reduction in antibody signal.

• Recombinant protein detection: Test antibody against recombinant ACAD11 protein expressed in systems such as E. coli using vectors like pET-21a(+) with E.coli codon bias in the first 100 base pairs .

What are emerging areas of research involving ACAD11 antibodies?

Several promising research directions are emerging for ACAD11 studies:

• Brain region-specific metabolism: Investigating how ACAD11 and other ACADs contribute to specialized fatty acid metabolism in different brain regions, potentially linking to neurological disorders .

• Non-energy metabolic functions: Exploring ACAD11's potential role in controlling fatty acid composition or catabolizing functional intermediates of aromatic amino acids in the central nervous system, rather than energy production .

• Peroxisomal vs. mitochondrial functions: Investigating the dual targeting of ACAD11 to both organelles given its PTS1 domain, and how this affects cellular metabolism.

• Alternative transcripts and domain-specific functions: Studying the significance of ACAD11's complex gene structure and potential alternative transcripts in different tissues .

What methodological advances may improve ACAD11 research beyond current antibody limitations?

Future methodological improvements may include:

• Domain-specific antibodies: Development of antibodies specifically targeting the APH domain versus the ACAD domain to study distinct functions.

• Live-cell imaging tools: Development of fluorescently tagged antibody fragments or nanobodies for live-cell tracking of ACAD11.

• Multiplex detection systems: Creation of antibody panels allowing simultaneous detection of multiple ACAD family members in the same sample.

• Spatial transcriptomics integration: Combining antibody-based protein detection with spatial transcriptomics to correlate protein localization with transcript expression patterns in complex tissues like brain.

• CRISPR-engineered cellular models: Development of endogenously tagged ACAD11 cell lines using CRISPR/Cas9 to study native protein expression without antibody limitations.