ACADL Antibody

What is ACADL and what is its biological significance?

ACADL (Long-chain specific acyl-CoA dehydrogenase, mitochondrial) is also known as LCAD and belongs to the acyl-CoA dehydrogenase family. It catalyzes the first reaction of the mitochondrial β-oxidation of fatty acids and is synthesized in the cytosol as a precursor that is larger than its mature form . This enzyme plays a critical role in energy metabolism, particularly in tissues with high energy demands such as muscle and heart, where long-chain fatty acids serve as important energy sources . Recent studies have also revealed that ACADL may have significant functions in the brain and other tissues that don't primarily rely on fat for energy generation .

What are the validated applications for ACADL antibodies?

ACADL antibodies have been validated for multiple research applications based on extensive testing. The primary applications include:

Additionally, positive Western blot detection has been confirmed in mouse kidney tissue, HepG2 cells, rat kidney tissue, HeLa cells, HEK-293 cells, and NIH/3T3 cells .

What are the recommended dilutions for ACADL antibodies in different applications?

Optimal dilutions for ACADL antibodies vary by application:

It is strongly recommended that researchers titrate these antibodies in each testing system to obtain optimal results as performance can be sample-dependent .

What are the molecular characteristics of ACADL protein?

The molecular characteristics of ACADL are well-documented:

This information is critical when validating antibody specificity through techniques like Western blotting .

What are the optimal antigen retrieval conditions when using ACADL antibodies for IHC?

For optimal immunohistochemistry results with ACADL antibodies, the following antigen retrieval conditions are recommended:

• Primary suggestion: Antigen retrieval with TE buffer pH 9.0

• Alternative method: Antigen retrieval with citrate buffer pH 6.0

These conditions have been specifically tested with human liver cancer tissue and human liver tissue samples . It is advisable to compare both methods when establishing protocols for new tissue types as antigen accessibility can vary significantly between tissues.

How can researchers validate ACADL antibody specificity?

Validating antibody specificity is critical for reliable research results. For ACADL antibodies, a multi-method approach is recommended:

• Western blot analysis: Compare observed band size (45-48 kDa) with the calculated molecular weight (48 kDa)

• Positive and negative controls: Use tissues known to express ACADL (kidney, liver) versus low-expressing tissues

• Knockout/knockdown validation: When possible, use CRISPR/Cas9 ACADL knockout cells as a negative control, similar to the approach used in ACADL functional studies

• Competitive blocking: Pre-incubate the antibody with purified ACADL protein before immunostaining to confirm binding specificity

• Cross-reactivity testing: Test against related proteins in the acyl-CoA dehydrogenase family to ensure specificity

Importantly, researchers should document that their antibody recognizes both the precursor and mature forms of the protein when studying tissues where processing may vary .

How should protein samples be prepared for optimal ACADL detection?

For reliable ACADL detection in various sample types:

• Tissue samples: Freshly harvested tissues should be immediately processed or flash-frozen in liquid nitrogen to preserve protein integrity

• Cell lysates: Use PBS with 0.02% sodium azide and appropriate protease inhibitors to prevent degradation

• Storage conditions: Maintain antibody at -20°C with 50% glycerol pH 7.3; stable for one year after shipment

• Mass spectrometry preparation: For proteomic analysis of ACADL, nanoLC-MS/MS analysis using a nanoACQUITY Ultra-Performance-LC coupled to a TripleTOF 5600 mass spectrometer has been successfully employed

• Immunoprecipitation: When performing IP-MS experiments to identify ACADL interactions, purification by antigen affinity methods has shown superior results

How are ACADL antibodies used to study cancer biology?

ACADL antibodies have become valuable tools in cancer research, particularly in understanding metabolic reprogramming in tumors:

• NSCLC research: In non-small cell lung cancer studies, ACADL antibodies have been used to analyze ACADL expression levels and correlate them with patient survival data from TCGA databases

• Expression analysis: Researchers have used ACADL antibodies to compare expression between tumor and normal tissues through:

  • Western blot quantification

  • Immunohistochemical staining

  • Proteomic analysis

• Mechanistic studies: ACADL antibodies have enabled investigation of the ACADL-YAP axis, revealing that ACADL regulates YAP phosphorylation levels and cellular localization, which influences cancer cell proliferation, invasion, and apoptosis

• In vivo validation: ACADL antibodies have been used to confirm protein expression in xenograft models, validating in vitro findings and establishing the tumorigenic effects of ACADL in NSCLC cells

The dual role of ACADL in cancer is particularly intriguing, as it appears to have different effects (inhibiting or promoting) depending on the tumor type .

What methods are available for studying ACADL in metabolic pathway research?

When investigating ACADL's role in metabolic pathways, researchers can employ several approaches:

• Transcriptional analysis: Real-time PCR with primer pairs targeted to unique exon junctions can measure expression levels of alternative ACADL transcripts across different tissues

• Enzyme activity assays: ACADL activity toward different substrates (particularly long-chain fatty acids with carbon chain lengths between C20-C26) can be assessed using spectrophotometric methods

• Substrate specificity analysis: ACADL shows optimal activity towards C22CoA, which is important when designing experiments to study its function

• Combined analysis: For comprehensive understanding of fatty acid oxidation, researchers should consider analyzing ACADL together with ACAD9 and ACAD11, which collectively accommodate the full spectrum of long-chain fatty acid substrates in mitochondrial β-oxidation

• Brain tissue studies: As ACADL has significant expression in human brain, specialized extraction protocols may be necessary when studying neurological tissues compared to more commonly studied tissues like liver or muscle

How can researchers enhance ACADL antibody performance through structural optimization?

Recent advances in antibody engineering provide several strategies for optimizing ACADL antibodies:

• Deep learning approaches: Tools like DeepAb can predict antibody Fv structure directly from sequence, allowing for rational design of optimized variants

• Deep mutational scanning (DMS): Experimental data from single-point mutations can be combined with computational methods to identify potentially beneficial modifications

• High-throughput screening: Production and testing of 200+ antibody variants can identify those with enhanced properties. In one study, 91% of designed clones exhibited increased thermal stability and 94% showed improved affinity

• Stability assessment: Testing for thermal and colloidal stability parameters (Tonset, Tm, Tagg) alongside affinity measurements (KD) can identify optimized antibodies

• Developability profile maintenance: When optimizing antibodies, researchers should monitor for nonspecific binding, aggregation propensity, and self-association to ensure the favorable developability profile is retained

Advanced computational-experimental workflows that don't require prior knowledge of the antibody-antigen interface have shown success in affinity enhancement by 5-21 fold while simultaneously improving thermostability by >2.5°C .

What strategies exist for identifying protein targets of antibodies using mass spectrometry?

For researchers seeking to identify and characterize protein targets of antibodies:

• Protein target identification protocol:

  • Enrich adducted proteins using immunoprecipitation with specific antibodies

  • Process for mass spectrometric analysis

  • Analyze with nanoLC-MS/MS systems coupled to high-resolution mass spectrometers

  • Process raw data with converters like MSDataConverter in .mgf peak list format

  • Interpret MS/MS data using algorithms like MASCOT against relevant databases (UniProtKB/SwissProt)

• Database selection: When identifying proteins, use appropriate database subsets (e.g., human, rat, or bovine) from comprehensive databases like Uniref100

• False discovery rate calculation: Calculate and report false discovery rates for protein identification to ensure reliability

• Differential modifications: Allow for specific modifications during database searches, such as carbamidomethylation of cysteines and oxidation of methionines

This approach has been successfully used to identify specific protein targets in complex biological samples and can be adapted for ACADL-related studies .

How can researchers develop and validate machine learning models for antibody optimization?

Machine learning approaches for antibody optimization are increasingly valuable:

• Model development approach:

  • Use data-driven model design with expert-engineered features

  • Incorporate both sequence-based features and structural information when available

  • Apply 5-fold cross-validation to optimize hyperparameters and increase model regularization

  • Test performance on out-of-distribution (OOD) validation datasets to ensure generalizability

• Integration with experimental workflow:

  • Implement ML models like AbRFC (Antibody Random Forest Classifier) within experimental pipelines

  • Use predictions to guide wet lab screening with limited designs (less than 100 per round)

  • Conduct multiple rounds of validation to iteratively improve models

• Performance metrics:

  • Measure improvement in antibody affinity (successful models have achieved >1000-fold improved affinity)

  • Assess performance across different antibody-epitope interactions to determine model generalizability

• Complementary approaches:

  • Combine models with technologies like yeast or phage display libraries

  • Consider large language models (LLMs) pre-trained on protein and antibody sequence space

This methodological approach has proven effective for optimizing antibodies against challenging targets, including variants of rapidly evolving pathogens .

How should researchers design experiments to study ACADL function in different cell types?

When investigating ACADL function across different cell types:

• Gene manipulation strategies:

  • For ACADL overexpression: Utilize lentiviral vectors carrying the full-length open reading frame with appropriate MOI (~20)

  • For ACADL knockout: Implement CRISPR/Cas9 KO plasmids with lipofectamine 2000 transfection

  • For rescue experiments: Transfect pcDNA 3.1 plasmids containing the full-length ACADL open reading frame

• Cell culture conditions:

  • Culture cells in appropriate medium (e.g., RPMI-1640 supplemented with 10% FBS)

  • Maintain at 37°C with 5% CO2 atmosphere

  • For suspension studies, use ultra-low attachment plates

• Experimental timing:

  • Conduct follow-up experiments 48 hours post-transfection

  • For immunofluorescence analysis, seed cells at 300,000 cells/ml and fix after 24 hours

• Control selection:

  • Include proper scrambled controls for knockout experiments

  • Use empty vector controls for overexpression studies

  • Consider parental cell lines as additional controls

These design considerations ensure reliable and reproducible results when studying ACADL function in experimental systems.

What considerations are important when developing ELISA assays for ACADL detection?

When developing or optimizing ELISA assays for ACADL:

• Assay principle selection: ACADL ELISA kits typically apply the competitive enzyme immunoassay technique, utilizing monoclonal anti-ACADL antibodies and ACADL-HRP conjugates

• Protocol optimization:

  • Incubate samples and buffer with ACADL-HRP conjugate in pre-coated plates (typically one hour)

  • Decant and wash five times

  • Incubate with HRP enzyme substrate to form blue-colored complex

  • Add stop solution to turn solution yellow

  • Measure intensity spectrophotometrically at 450nm

• Interpretation considerations:

  • The intensity of color is inversely proportional to ACADL concentration

  • This occurs because ACADL from samples competes with ACADL-HRP conjugate for limited anti-ACADL antibody binding sites

• Standard curve development:

  • Plot the intensity of color (O.D.) against concentration of standards

  • Interpolate ACADL concentration in each sample from this standard curve

• Validation controls:

  • Include positive and negative controls

  • Prepare standard curves with recombinant ACADL protein

  • Validate across multiple sample types to ensure reliability

This methodological approach ensures accurate quantification of ACADL in research samples.