ACADSB Antibody

What is ACADSB and what is its biological function?

ACADSB (acyl-Coenzyme A dehydrogenase, short/branched chain) is a mitochondrial enzyme that plays a critical role in protein metabolism, specifically in the breakdown of branched-chain amino acids. The enzyme is particularly important in processing isoleucine, where it catalyzes the third step in the metabolic pathway by converting 2-methylbutyryl-CoA to tiglyl-CoA through an oxidation reaction .

ACADSB is localized within mitochondria throughout the body's cells and contributes to energy production by helping convert amino acids into forms that can be utilized for cellular energy. Mutations in the ACADSB gene can lead to short/branched chain acyl-CoA dehydrogenase (SBCAD) deficiency, a condition where the body cannot properly break down isoleucine .

What are the recommended applications for ACADSB antibodies in research?

ACADSB antibodies have been validated for multiple research applications:

It is strongly recommended that each antibody be titrated in your specific experimental system to obtain optimal results, as sample-dependent variations can affect performance . For IHC applications, antigen retrieval with TE buffer (pH 9.0) is suggested, though citrate buffer (pH 6.0) may also be used as an alternative .

What is the molecular weight of ACADSB protein, and how is this relevant for antibody validation?

The ACADSB protein has a calculated molecular weight of 47 kDa (432 amino acids), but is typically observed at 45-47 kDa in experimental conditions . This information is crucial for antibody validation by Western blot, as it allows researchers to confirm target specificity by comparing the observed band to the expected molecular weight.

When validating a new ACADSB antibody, always run appropriate positive controls such as mouse kidney tissue, HepG2 cells, or HEK-293 cells, which have been documented to express detectable levels of ACADSB protein .

What sample types are most suitable for ACADSB antibody detection?

Based on validation data, the following sample types have been successfully used with ACADSB antibodies:

• Tissue samples: Mouse kidney tissue, human breast cancer tissue

• Cell lines: HepG2 cells, HEK-293 cells

• Species reactivity: Most ACADSB antibodies show reactivity with human, mouse, and rat samples. Some antibodies have broader reactivity including bat, cow, dog, monkey, pig, and zebrafish (Danio rerio)

For optimal results in immunohistochemistry applications, human breast cancer tissue has been documented as a positive control .

How is ACADSB involved in ferroptosis regulation, and what are the methodological approaches to study this relationship?

ACADSB has been found to regulate ferroptosis, a form of iron-dependent cell death characterized by lipid peroxidation. Research has shown that ACADSB negatively regulates the expression of glutathione reductase and glutathione peroxidase 4 (GPX4), which are the main enzymes responsible for clearing glutathione (GSH) in colorectal cancer cells .

To study ACADSB's role in ferroptosis, researchers can employ the following methodological approaches:

• Overexpression and knockdown experiments: Manipulate ACADSB expression levels and assess markers of ferroptosis.

• Ferroptosis biomarker measurement: Measure malondialdehyde, Fe²⁺, superoxide dismutase, lipid peroxidation, and GSH levels.

• Mitochondrial function assessment: Since ACADSB is localized in mitochondria, assess mitochondrial lipid peroxidation and function.

Research has demonstrated that ACADSB overexpression enhances the concentration of malondialdehyde, Fe²⁺, superoxide dismutase, and lipid peroxidation, while reducing GSH concentration in colorectal cancer cells—all important indicators of ferroptosis .

What role does ACADSB play in cancer progression, and how can researchers experimentally evaluate this function?

ACADSB has been identified as having potential tumor suppressor functions in colorectal cancer (CRC). Studies have found that:

To experimentally evaluate ACADSB's role in cancer progression, researchers can:

• Compare expression levels: Analyze ACADSB expression in tumor vs. normal tissues using IHC, WB, or qPCR

• Conduct gain/loss-of-function studies: Overexpress or knock down ACADSB in cancer cell lines and assess:

  • Cell migration (wound healing assay, transwell migration assay)

  • Invasion (matrigel invasion assay)

  • Proliferation (MTT/CCK-8 assay, EdU incorporation)

  • Colony formation ability

• Investigate mechanism: Analyze ferroptosis markers and pathways influenced by ACADSB manipulation

• In vivo studies: Use xenograft models with ACADSB-modulated cells to assess tumor growth and metastasis

These approaches can help elucidate how ACADSB affects cancer progression through regulation of ferroptosis and potentially other mechanisms .

What are the optimal conditions for ACADSB antibody validation, and how should researchers address cross-reactivity concerns?

For rigorous ACADSB antibody validation, researchers should implement the following protocol:

• Western Blot validation:

  • Use fresh protein extracts from known positive samples (mouse kidney tissue, HepG2 cells, HEK-293 cells)

  • Include negative controls (tissues/cells with confirmed low ACADSB expression)

  • Look for specific bands at 45-47 kDa

  • Recommended dilution range: 1:200-1:1000

  • Confirm specificity through ACADSB knockdown/knockout lysates

• IHC validation:

  • Test multiple antigen retrieval methods (TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative)

  • Use positive control tissues (human breast cancer tissue)

  • Recommended dilution range: 1:50-1:500

  • Include isotype controls to assess non-specific binding

• Cross-reactivity assessment:

  • Test antibody across multiple species when working with animal models

  • Verify antibody specificity using overexpression systems

  • For polyclonal antibodies, consider pre-absorption with target antigen to confirm specificity

  • Examine potential cross-reactivity with other acyl-CoA dehydrogenase family members

• Epitope considerations:

  • For reproducible results, select antibodies recognizing conserved epitopes when working across species

  • Consider antibodies raised against different protein regions for confirmation of results

How do mutations in ACADSB affect protein function and what methodologies can detect these functional changes?

Mutations in the ACADSB gene can lead to short/branched chain acyl-CoA dehydrogenase (SBCAD) deficiency. More than 10 ACADSB gene mutations have been identified, with the following effects:

• Amino acid substitutions that affect protein function

• Truncated enzymes missing several amino acids

• Reduced or absent enzymatic activity

• Impaired isoleucine metabolism

To detect and characterize these functional changes, researchers can employ:

• Enzymatic activity assays:

  • Measure SBCAD activity in cell/tissue extracts using 2-methylbutyryl-CoA as substrate

  • Monitor conversion to tiglyl-CoA through spectrophotometric or LC-MS methods

• Metabolite profiling:

  • Analyze 2-methylbutyrylglycine levels in urine or 2-methylbutyrylcarnitine in plasma/blood

  • Use GC-MS or LC-MS/MS for metabolite detection and quantification

• Protein stability assessment:

  • Pulse-chase experiments to evaluate protein half-life

  • Circular dichroism to assess structural changes

  • Thermal shift assays to evaluate protein stability

• Subcellular localization studies:

  • Immunofluorescence microscopy with mitochondrial markers

  • Cell fractionation followed by Western blot analysis

• Computational approaches:

  • Structural modeling to predict mutation impact

  • Molecular dynamics simulations to assess functional consequences

These methodologies help understand how ACADSB mutations affect protein function and contribute to disease phenotypes .

What are the recent advances in understanding ACADSB's role in iron homeostasis and how can researchers investigate this relationship?

Recent research has begun to uncover connections between ACADSB, ferroptosis, and iron homeostasis. Iron plays a critical role in ferroptosis, a form of cell death characterized by iron-dependent lipid peroxidation . Since ACADSB has been found to regulate ferroptosis in colorectal cancer cells, this suggests potential involvement in iron metabolism.

To investigate the relationship between ACADSB and iron homeostasis, researchers can:

• Measure iron-related parameters upon ACADSB manipulation:

  • Quantify intracellular Fe²⁺ levels using fluorescent indicators (e.g., Phen Green SK)

  • Assess labile iron pool using iron chelators

  • Measure expression of iron transport and storage proteins (ferritin, transferrin receptor, ferroportin)

• Investigate regulatory relationships:

  • Determine whether iron levels affect ACADSB expression or activity

  • Assess whether ACADSB affects the expression of iron regulatory proteins (IRPs) or iron responsive elements (IREs)

  • Examine the impact of ACADSB on mitochondrial iron uptake and utilization

• Explore signaling pathways:

  • Investigate interactions with known iron metabolism regulators

  • Assess how ACADSB affects the p53 pathway, which has been found to promote ferroptosis through transcription-dependent mechanisms

  • Examine potential involvement in reactive oxygen species generation within mitochondria

• Therapeutic implications:

  • Test iron chelators or ferroptosis inhibitors in ACADSB-overexpressing or depleted models

  • Evaluate combination approaches targeting both ACADSB and iron metabolism for potential therapeutic applications

These approaches can help elucidate the emerging role of ACADSB in iron homeostasis and its implications for disease pathogenesis and treatment .

What are common issues encountered with ACADSB antibodies in Western blot applications and how can they be resolved?

Researchers working with ACADSB antibodies may encounter several technical challenges in Western blot applications:

For optimal results, follow the specific protocol recommended for ACADSB antibody , ensure proper sample preparation, and validate results with appropriate positive and negative controls.

How can researchers optimize ACADSB antibody use in immunohistochemistry for different tissue types?

Optimizing ACADSB antibody use in immunohistochemistry requires tissue-specific considerations:

• Antigen retrieval optimization:

  • For most tissues, TE buffer (pH 9.0) is recommended for ACADSB detection

  • For challenging tissues, test alternative methods like citrate buffer (pH 6.0)

  • Optimize retrieval time and temperature based on tissue type

• Tissue-specific dilution determination:

  • Start with the recommended range (1:50-1:500)

  • Perform titration experiments to determine optimal concentration for each tissue type

  • For tissues with high endogenous ACADSB expression, use higher dilutions (1:200-1:500)

  • For tissues with lower expression, use lower dilutions (1:50-1:100)

• Fixation considerations:

  • Optimal fixation time varies by tissue thickness

  • Overfixation can mask epitopes—consider extended antigen retrieval for heavily fixed samples

  • For fatty tissues, ensure complete fixation penetration

• Tissue-specific controls:

  • For human samples, breast cancer tissue has been validated as positive control

  • For mouse studies, kidney tissue shows reliable ACADSB expression

  • Always include tissue-matched negative controls using isotype antibodies

• Signal amplification strategies:

  • For tissues with low ACADSB expression, consider using polymer-based detection systems

  • For dual staining applications, select compatible chromogens based on tissue autofluorescence/pigmentation

Following these optimization strategies will help ensure specific and reproducible ACADSB detection across different tissue types.

How can ACADSB antibodies be used to investigate mitochondrial dysfunction in metabolic diseases?

ACADSB antibodies provide valuable tools for investigating mitochondrial dysfunction in metabolic diseases through several methodological approaches:

• Colocalization studies:

  • Perform double immunofluorescence with ACADSB antibodies and mitochondrial markers (TOM20, COXIV)

  • Quantify colocalization coefficients to assess mitochondrial localization efficiency

  • Compare healthy vs. diseased tissues to identify alterations in subcellular distribution

• Mitochondrial fraction analysis:

  • Isolate mitochondria from tissues/cells of interest

  • Use ACADSB antibodies to quantify protein levels in mitochondrial fractions by Western blot

  • Compare ACADSB levels across different metabolic states or disease models

• Functional correlation studies:

  • Measure ACADSB protein levels in relation to:

    • Mitochondrial respiratory complex activities

    • Lipid peroxidation markers

    • Isoleucine metabolism intermediates

  • Correlate expression patterns with clinical parameters in metabolic disease cohorts

• Dynamic regulation assessment:

  • Monitor ACADSB expression changes upon metabolic stress (nutrient deprivation, high-fat diet)

  • Investigate post-translational modifications of ACADSB using phospho-specific antibodies

  • Examine protein-protein interactions with other mitochondrial enzymes

• Therapeutic response monitoring:

  • Use ACADSB antibodies to assess treatment effects on protein expression/localization

  • Evaluate mitochondrial adaptation to metabolic interventions

These approaches allow researchers to gain mechanistic insights into how ACADSB dysfunction contributes to mitochondrial impairment in metabolic diseases, potentially identifying new therapeutic targets .

What methodologies can researchers use to investigate the relationship between ACADSB and p53 in cancer models?

The relationship between ACADSB and p53 in cancer models can be investigated using several strategic approaches:

• Expression correlation analysis:

  • Perform dual immunostaining for ACADSB and p53 in cancer tissue microarrays

  • Quantify expression correlation using digital pathology tools

  • Analyze public datasets (TCGA, GEO) for correlation between ACADSB and TP53 gene expression

• Mechanistic pathway investigation:

  • Modulate p53 status (activation with Nutlin-3a, knockdown, or knockout) and assess ACADSB expression

  • Conversely, manipulate ACADSB levels and monitor effects on p53 activity

  • Identify potential p53 binding sites in the ACADSB promoter through ChIP assays

  • Perform luciferase reporter assays to validate transcriptional regulation

• Ferroptosis pathway analysis:

  • Since p53 has been found to promote ferroptosis in a transcription-dependent manner :

    • Assess whether ACADSB is a transcriptional target of p53

    • Measure ferroptosis markers after p53 activation in ACADSB-deficient vs. normal cells

    • Determine if ACADSB is required for p53-mediated ferroptosis

• Therapeutic response studies:

  • Evaluate cancer cell sensitivity to ferroptosis inducers based on ACADSB and p53 status

  • Test combination approaches targeting both pathways

  • Develop p53/ACADSB status as potential predictive biomarkers for therapy response

• In vivo model validation:

  • Generate xenograft models with modulated ACADSB and p53 expression

  • Assess tumor growth, metastasis, and response to therapy

  • Analyze tissue samples for markers of ferroptosis and pathway activation

These methodologies will help elucidate the functional relationship between ACADSB and p53, potentially identifying new therapeutic strategies for cancer treatment .

What emerging technologies can enhance ACADSB antibody-based research?

Several cutting-edge technologies are poised to advance ACADSB antibody-based research:

• Single-cell antibody techniques:

  • Single-cell Western blotting to analyze ACADSB expression heterogeneity

  • Mass cytometry (CyTOF) incorporating ACADSB antibodies for high-dimensional analysis

  • Microfluidic antibody capture for rare cell population analysis

• Advanced imaging applications:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization of ACADSB within mitochondria

  • Expansion microscopy to visualize ACADSB distribution with enhanced resolution

  • Live-cell imaging with split fluorescent protein systems to monitor ACADSB dynamics

• Spatial transcriptomics integration:

  • Combining ACADSB antibody staining with spatial transcriptomics for correlative protein-RNA analysis

  • Multiplexed ion beam imaging (MIBI) for simultaneous detection of ACADSB and multiple markers

  • Digital spatial profiling to quantify ACADSB expression in specific tissue regions

• Antibody engineering advances:

  • Nanobodies against ACADSB for improved tissue penetration and resolution

  • Bispecific antibodies targeting ACADSB and other ferroptosis markers simultaneously

  • Recombinant antibody fragments optimized for specific applications

• High-throughput applications:

  • Antibody microarrays for profiling ACADSB interactions

  • Automated IHC platforms for large-scale clinical sample analysis

  • Machine learning algorithms for quantitative analysis of ACADSB expression patterns

These technological advances will enable more precise, comprehensive, and efficient investigation of ACADSB's role in normal physiology and disease states.

How might understanding ACADSB's role in ferroptosis contribute to developing new therapeutic strategies?

Understanding ACADSB's role in ferroptosis opens several promising avenues for therapeutic development:

• Targeted cancer therapies:

  • Since ACADSB regulates ferroptosis and is downregulated in colorectal cancer :

    • Develop small molecules to enhance ACADSB expression/activity

    • Design combination approaches targeting ACADSB and ferroptosis pathways

    • Stratify patients based on ACADSB expression for ferroptosis-inducing therapies

• Metabolic disease interventions:

  • For conditions related to ACADSB deficiency or dysfunction:

    • Design enzyme replacement therapies

    • Develop chaperones to stabilize mutant ACADSB proteins

    • Explore metabolic bypass strategies to compensate for impaired isoleucine metabolism

• Ferroptosis modulation approaches:

  • For conditions where ferroptosis contributes to pathology:

    • Target ACADSB to inhibit excessive ferroptosis

    • For cancer therapy, enhance ACADSB activity to promote ferroptosis in tumor cells

    • Develop biomarkers based on ACADSB status to predict ferroptosis susceptibility

• Mitochondrial protection strategies:

  • Since ACADSB is mitochondrial and linked to ferroptosis:

    • Design mitochondria-targeted antioxidants based on ACADSB interactions

    • Develop approaches to modulate ACADSB-dependent lipid metabolism

    • Create mitochondrial-targeted delivery systems for ACADSB modulators

• Precision medicine applications:

  • Utilize ACADSB expression/mutation status as:

    • Prognostic biomarkers in cancer

    • Predictors of response to ferroptosis-inducing therapies

    • Guides for personalized treatment selection