ADSL Antibody

What is ADSL and what is its role in cellular metabolism?

ADSL (Adenylosuccinate Lyase) is a member of the Lyase 1 protein family that plays a critical role in purine metabolism. In humans, the canonical protein consists of 484 amino acid residues with a molecular mass of 54.9 kDa . ADSL catalyzes two non-sequential steps in de novo AMP synthesis: it converts (S)-2-(5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido)succinate (SAICAR) to fumarate plus 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide, contributing to de novo IMP synthesis; it also converts succinyladenosine monophosphate (SAMP) to AMP and fumarate . These reactions are essential for DNA replication and repair processes .

The enzyme is ubiquitously expressed across many tissue types, emphasizing its fundamental importance in cellular metabolism . Two different isoforms have been reported for this protein, allowing for potential tissue-specific regulation of purine metabolism .

How does ADSL function in different model organisms?

ADSL is highly conserved across species, with orthologs reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . This conservation underscores the evolutionary importance of purine metabolism. Research models utilizing these species have provided valuable insights into ADSL function and pathology.

In zebrafish and chicken embryos, ADSL deficiency results in impaired neurogenesis and microcephaly, demonstrating its critical role in neurodevelopment . Specifically, ADSL-deficient zebrafish embryos display neuroprogenitor attrition, which can be rescued by pharmacological inhibition of de novo purine synthesis (DNPS), suggesting a complex relationship between purine metabolism and neural development .

What are the known structural characteristics of human ADSL protein?

Human ADSL protein has the following key structural characteristics:

The structural integrity of ADSL is essential for its catalytic function. Mutations affecting protein folding or catalytic sites can lead to ADSL deficiency disorders with varying degrees of severity .

What are the primary applications for ADSL antibodies in research?

ADSL antibodies are versatile research tools with multiple applications in molecular and cellular biology:

When selecting an antibody for a specific application, researchers should consider factors such as host species, clonality, epitope specificity, and detection method compatibility .

How should researchers optimize Western blot protocols for ADSL detection?

For optimal Western blot detection of ADSL:

• Sample preparation: Use appropriate lysis buffers with protease inhibitors to prevent degradation of the 54.9 kDa ADSL protein.

• Gel percentage selection: A 10% SDS-PAGE gel typically provides good separation for the ADSL protein.

• Primary antibody selection: Choose antibodies that recognize endogenous levels of ADSL protein, such as polyclonal antibodies raised against full-length ADSL or specific domains .

• Controls: Include positive controls (tissues/cells known to express ADSL) and negative controls (knockdown/knockout samples if available) to validate specificity.

• Blocking optimization: Typically, 5% non-fat milk or BSA in TBST provides effective blocking, but optimization may be required based on the specific antibody.

• Validation approach: When possible, confirm results using multiple antibodies targeting different epitopes of ADSL to ensure specificity .

What considerations are important when selecting an ADSL antibody?

When selecting an ADSL antibody, researchers should consider:

• Epitope specificity: Antibodies targeting different regions (e.g., AA 1-310, AA 188-237) may have different recognition properties . Consider the region of interest for your study, especially if focusing on specific domains or isoforms.

• Species reactivity: Many ADSL antibodies react with human, mouse, and rat ADSL, but cross-reactivity with other species varies . Verify reactivity with your experimental model.

• Clonality: Polyclonal antibodies generally provide broader epitope recognition, while monoclonal antibodies offer higher specificity for a single epitope .

• Validation data: Review available data demonstrating antibody specificity and performance in your application of interest.

• Conjugation: Consider whether an unconjugated antibody or one conjugated to a reporter molecule (e.g., fluorophore, enzyme) is more appropriate for your experiment .

How does ADSL deficiency impact neurodevelopment?

ADSL deficiency (ADSLD) manifests as a spectrum of neurodevelopmental disorders with significant clinical implications:

• Clinical manifestations: ADSLD causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder .

• Biochemical profile: Patients typically maintain normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, particularly S-Ado and SAICAr .

• Neurotoxic mechanisms: SAICAr accumulation is implicated in neurotoxic effects, though the precise mechanisms remain under investigation .

• Developmental impacts: In model organisms, ADSL deficiency leads to impaired neurogenesis and microcephaly, suggesting critical roles in neural progenitor development and maintenance .

What cellular pathways are disrupted in ADSL deficiency?

ADSL deficiency disrupts multiple cellular pathways:

ADSL-depleted cells frequently lack Ki67 expression (indicating cell cycle exit), show increased p53 levels, and accumulate in G1 phase . Deletion of TP53 rescues proliferation and prevents G1 arrest, suggesting p53-dependent mechanisms of cell cycle regulation in response to ADSL deficiency .

How can researchers experimentally differentiate between purine depletion and metabolite accumulation effects?

Distinguishing between these mechanisms is crucial for understanding ADSL deficiency pathology:

• Nucleoside supplementation: Adding adenosine or a nucleoside mix can rescue phenotypes resulting from purine depletion. Research shows this approach reduces DNA damage markers like 53BP1 foci and γH2AX in ADSL-depleted cells .

• PAICS inhibition: Using inhibitors like MRT00252040 to block upstream reactions in de novo purine synthesis prevents SAICAr accumulation without restoring purine levels. This approach does not rescue DNA damage phenotypes, suggesting these are primarily due to purine depletion rather than metabolite accumulation .

• Rescue experiments: In zebrafish embryos, neuroprogenitor attrition was rescued by pharmacological inhibition of de novo purine synthesis but not by increased nucleotide concentration, highlighting the complexity of pathway-specific effects .

• SAICAr administration: Directly applying SAICAr to normal cells can help identify which phenotypes are specifically caused by this metabolite. Research shows that primary ciliogenesis is impaired specifically by SAICAr accumulation .

What is the optimal method for quantifying ADSL protein in biological samples?

The optimal method depends on the specific research question and sample type:

• ELISA: For precise quantification in serum, plasma, or cell culture supernatants, sandwich ELISA provides high sensitivity (<0.188 ng/ml) with a detection range of 0.313-20 ng/ml . This is particularly useful for clinical samples or when comparing ADSL levels across conditions.

• Western blot: For relative quantification and molecular weight confirmation, western blotting using validated antibodies (such as rabbit polyclonal antibodies purified by immunogen affinity chromatography) provides reliable results . Always include appropriate loading controls and standard curves.

• Immunohistochemistry/Immunofluorescence: For spatial localization within tissues or cells, these techniques provide valuable information about ADSL distribution patterns .

• Mass spectrometry: For absolute quantification and identification of post-translational modifications, targeted proteomics approaches can complement antibody-based methods.

How can researchers study ADSL function in cellular models?

Multiple approaches exist for studying ADSL function in cellular systems:

• Gene silencing: siRNA or shRNA approaches targeting ADSL can reveal phenotypes associated with reduced enzyme activity. This has been used to demonstrate effects on cell proliferation, DNA damage, and primary ciliogenesis .

• CRISPR/Cas9 gene editing: For complete knockout or introduction of specific mutations mimicking those found in ADSL deficiency patients.

• Rescue experiments: Reintroduction of wild-type ADSL (such as siRNA-resistant alleles) can confirm specificity of observed phenotypes .

• Metabolite profiling: Analysis of purine metabolites and ADSL substrates can provide biochemical confirmation of enzyme dysfunction.

• Cell cycle analysis: Flow cytometry and Ki67 staining can reveal effects on proliferation and cell cycle progression .

What approaches can identify protein interaction partners of ADSL?

Understanding ADSL's interaction network can provide insights into its regulation and additional functions:

• Co-immunoprecipitation: Using ADSL antibodies to pull down the protein complex, followed by mass spectrometry or western blotting for known candidates.

• Proximity labeling: BioID or APEX approaches fused to ADSL can identify proteins in close proximity within living cells.

• Yeast two-hybrid screening: Can identify direct protein-protein interactions, though requires confirmation in mammalian systems.

• Cross-linking mass spectrometry: Provides structural insights into protein complexes involving ADSL.

• Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC): Can confirm specific interactions and provide spatial information in living cells.

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

Researchers may encounter several challenges when working with ADSL antibodies:

• Non-specific binding: Optimize blocking conditions and antibody concentration. Consider using antibodies purified by immunogen affinity chromatography .

• Isoform specificity: Choose antibodies targeting regions common to all isoforms for total ADSL detection, or select epitopes unique to specific isoforms when discrimination is desired .

• Cross-reactivity with related proteins: Validate specificity using ADSL knockdown/knockout controls or recombinant protein competition assays.

• Batch-to-batch variability: Monoclonal antibodies generally provide more consistent results across batches compared to polyclonals .

• Fixation sensitivity for IHC/IF: Test multiple fixation methods if initial attempts yield poor results, as ADSL epitopes may be sensitive to specific fixatives.

How should researchers validate the specificity of ADSL antibodies?

A multi-faceted validation approach is recommended:

• Positive and negative controls: Include tissues/cells with known ADSL expression patterns and, when possible, ADSL-deficient samples.

• Recombinant protein controls: Use purified ADSL protein as a positive control in western blots.

• siRNA/shRNA knockdown: Demonstrate reduced signal following ADSL depletion.

• Multiple antibodies: When possible, confirm findings using antibodies targeting different epitopes .

• Mass spectrometry validation: For novel applications or tissues, confirm antibody specificity by immunoprecipitation followed by mass spectrometry.

• Genetic models: When available, use ADSL knockout/knockdown models as definitive negative controls.

What quality control metrics should be considered when evaluating ADSL detection results?

When evaluating experimental results:

• Signal-to-noise ratio: Assess background levels and ensure specific signal is clearly distinguishable.

• Molecular weight confirmation: ADSL should appear at approximately 54.9 kDa in western blots .

• Reproducibility: Results should be consistent across technical and biological replicates.

• Antibody concentration optimization: Titrate antibodies to determine optimal working concentration for each application.

• Comparison with known expression patterns: Results should align with established ADSL expression patterns across tissues and subcellular locations.

• Appropriate controls: Include loading controls for western blots, isotype controls for flow cytometry, and secondary-only controls for immunostaining.

How can ADSL antibodies contribute to understanding ciliopathies?

Recent research has uncovered connections between ADSL and ciliogenesis:

• Phenotypic overlap: ADSL-deficient zebrafish display phenotypes commonly linked to ciliopathies, suggesting a mechanistic connection .

• SAICAr effects: Primary ciliogenesis is impaired specifically by loss of ADSL or administration of SAICAr, implicating this metabolite in cilia-related pathologies .

• Research applications: ADSL antibodies can be used to study co-localization with ciliary markers, examine protein levels in ciliopathy models, and investigate potential interactions with ciliary proteins.

• Therapeutic implications: Understanding the relationship between ADSL and ciliogenesis could lead to novel therapeutic approaches for both ADSL deficiency and ciliopathies.

What role might ADSL play in DNA damage response pathways?

ADSL deficiency has significant impacts on genome stability:

• Replication stress: ADSL-depleted cells show increased chromatin-bound RPA levels, indicating single-stranded DNA accumulation .

• Double-strand breaks: Increased 53BP1 foci and γH2AX levels in ADSL-depleted cells indicate DNA double-strand break formation .

• ATM signaling: DNA damage in ADSL-depleted cells involves active ATM signaling, as 53BP1 foci are reduced by ATM inhibition .

• Purine dependency: Nucleoside supplementation rescues DNA damage, implicating purine depletion rather than metabolite accumulation in this phenotype .