ADSL Antibody

What is ADSL protein and why is it important in research?

Adenylosuccinate lyase (ADSL) is a 484 amino acid protein (54.9 kDa) that plays crucial roles in de novo purine synthesis (DNPS) and the purine nucleotide cycle. It catalyzes two non-sequential reactions in AMP biosynthesis: the conversion of SAICAR to fumarate plus AICAR (contributing to IMP synthesis), and the conversion of succinyladenosine monophosphate (SAMP) to AMP and fumarate . ADSL is particularly significant in neurodevelopmental research as ADSL deficiency causes several pathologies including microcephaly and autism spectrum disorder . As a ubiquitously expressed enzyme, ADSL is essential for maintaining cellular energy levels and nucleotide synthesis .

How do I select the most appropriate ADSL antibody for my experiment?

Selection should be based on:

• Validated Applications: Choose antibodies specifically validated for your intended application (WB, IHC, IF, ELISA)

• Species Reactivity: Ensure the antibody recognizes ADSL in your model organism (human, mouse, rat, etc.)

• Antibody Type: Consider polyclonal for higher sensitivity or monoclonal for greater specificity

• Citation Records: Prioritize antibodies with published validation in peer-reviewed literature

• Epitope Recognition: For domain-specific studies, select antibodies targeting specific regions (e.g., AA 1-310 vs. full-length)

The top validated ADSL antibodies based on citation frequency include Atlas Antibodies HPA000525 (6 references), Proteintech Group 15264-1-AP (3 references), and Novus Biologicals NBP1-87406 (2 references) .

What are the key differences between polyclonal and monoclonal ADSL antibodies?

How should I optimize Western blot conditions for ADSL detection?

For optimal Western blot detection of ADSL:

• Sample Preparation:

  • Use RIPA buffer with protease inhibitors for efficient extraction

  • Load 20-30 μg of total protein per lane

  • Include positive controls such as human cell lines (RPE-1)

• Electrophoresis and Transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution of the 54.9 kDa ADSL protein

  • Transfer to PVDF membranes at 100V for 1 hour or 30V overnight

• Antibody Incubation:

  • Primary antibody: Use validated ADSL antibodies at manufacturer-recommended dilutions (typically 1:1000)

  • Secondary antibody: HRP-conjugated anti-species antibodies (1:5000-1:10000)

  • Include proper controls (loading control, no primary antibody)

• Detection:

  • Use enhanced chemiluminescence (ECL) for standard detection

  • For quantitative analysis, consider fluorescently labeled secondary antibodies

  • Expected band size: ~55 kDa

How can I effectively use ADSL antibodies for immunocytochemistry?

For successful immunocytochemistry with ADSL antibodies:

• Cell Preparation:

  • Culture cells on coverslips to 70-80% confluence

  • Fix cells with 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 0.1% Triton X-100 (5 minutes)

• Blocking and Antibody Incubation:

  • Block with 5% BSA or normal serum (1 hour)

  • Incubate with primary ADSL antibody (1:100 dilution for IF applications)

  • Wash thoroughly (3x5 minutes with PBS)

  • Incubate with fluorophore-conjugated secondary antibody (1:200-1:500)

• Imaging Considerations:

  • ADSL typically shows cytoplasmic localization

  • Co-stain with organelle markers to confirm subcellular distribution

  • Include DAPI for nuclear visualization

• Controls:

  • Include ADSL-depleted cells (siRNA-treated) as negative controls

  • Consider using ARL13B antibody (1:100) as a marker for primary cilia when studying ADSL in relation to ciliogenesis

What are the most sensitive detection methods for quantifying ADSL using antibodies?

The following methods are arranged by increasing sensitivity:

• Western Blot: Detects endogenous ADSL levels, semi-quantitative

  • Sensitivity: ~1-10 ng of target protein

  • Best for relative quantification between samples

• Standard ELISA: More quantitative than Western blot

  • Sensitivity: ~0.1-1 ng/ml

  • Sandwich ELISA formats using capture and detection ADSL antibodies offer improved specificity

• Immunoprecipitation followed by Western blot (IP-WB):

  • Enables detection of protein interactions

  • Particularly useful for studying ADSL complex formation

  • Multiple antibodies available for IP applications, including Santa Cruz (C-11)

• Enhanced Chemiluminescence ELISA:

  • Sensitivity: ~1-10 pg/ml

  • HRP-based detection systems significantly improve sensitivity

• Immunochromatography:

  • Rapid detection method

  • Available with certain ADSL antibodies

  • Best for qualitative analysis

How can I validate the specificity of my ADSL antibody?

Comprehensive validation requires multiple approaches:

• Positive and Negative Controls:

  • Use ADSL-knockdown cells (siRNA-treated) as negative controls

  • Use recombinant ADSL protein as positive control

  • Compare recognition patterns across multiple tissues with known ADSL expression levels

• Multiple Detection Methods:

  • Cross-validate results using different applications (WB, IF, IHC)

  • Compare results from antibodies recognizing different ADSL epitopes

  • Verify the expected molecular weight (~55 kDa)

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide

  • Signal should be abolished or significantly reduced

• Knockout/Knockdown Validation:

  • Compare results in ADSL-deficient models versus controls

  • Signal should decrease proportionally to the reduction in ADSL levels

  • Consider using an siRNA-resistant ADSL construct (ADSL*) for rescue experiments

• Mass Spectrometry Confirmation:

  • Perform IP followed by mass spectrometry analysis

  • Confirms identity of the immunoprecipitated protein

I'm observing multiple bands in my Western blot with ADSL antibody. What could this indicate?

Multiple bands may result from:

• Isoforms: Up to 2 different isoforms of ADSL have been reported in humans , which could appear as distinct bands

  • Main band: ~55 kDa (canonical isoform, 484 amino acids)

  • Alternative splice variants may show different molecular weights

• Post-translational Modifications:

  • Phosphorylation, ubiquitination, or other modifications can alter migration

  • Consider phosphatase treatment to determine if phosphorylation contributes to band shifts

• Proteolytic Cleavage:

  • Sample degradation during preparation

  • Add additional protease inhibitors and maintain cold conditions during extraction

• Cross-reactivity:

  • Non-specific binding to related proteins (check sequence homology)

  • Test antibody on ADSL-depleted samples to identify non-specific bands

  • Consider antibodies targeting different epitopes to confirm specificity

• Antibody Quality Issues:

  • Batch variation (especially with polyclonal antibodies)

  • Try antibodies from different suppliers with validated applications

What controls should I include when using ADSL antibodies to study ADSL deficiency models?

For rigorous experimental design when studying ADSL deficiency:

• Positive Controls:

  • Samples with known ADSL expression

  • Recombinant ADSL protein standards

  • Cells transfected with ADSL expression constructs

• Negative Controls:

  • ADSL-depleted cells using validated siRNA

  • No primary antibody controls for IF/IHC

  • Isotype controls for flow cytometry applications

• Rescue Controls:

  • Re-expression of siRNA-resistant ADSL (ADSL*) in depleted cells

  • Should restore normal phenotype and antibody detection

• Pharmacological Controls:

  • Nucleoside supplementation to bypass purine synthesis defects

  • PAICS inhibitor (MRT00252040) to modulate upstream pathway steps

  • ATM inhibitor (KU5593) to examine DNA damage response relationship

• Pathway Validation:

  • Monitor downstream effects (e.g., DNA damage markers like 53BP1, γH2AX)

  • Examine purine metabolite levels using metabolomics approaches

How can ADSL antibodies be utilized to investigate neurodevelopmental disorders?

ADSL antibodies are valuable tools for studying ADSL deficiency-related neurodevelopmental disorders:

• Tissue Distribution Analysis:

  • Compare ADSL levels in normal vs. pathological brain tissues

  • Use IHC with ADSL antibodies to examine regional expression patterns

  • Correlate with neuronal markers to identify affected cell populations

• Animal Model Validation:

  • Confirm ADSL knockdown/knockout in chicken and zebrafish models

  • Analyze neuroprogenitor effects in developing embryos

  • Correlate ADSL levels with microcephaly phenotypes

• Pathway Analysis:

  • Investigate relationships between ADSL and DNA damage signaling using co-staining with 53BP1, γH2AX

  • Examine ADSL's role in primary ciliogenesis using ARL13B co-staining

  • Evaluate p53-dependent cell cycle exit mechanisms using Ki67 and p53 antibodies

• Therapeutic Development:

  • Screen compounds that rescue ADSL deficiency phenotypes

  • Monitor ADSL stability and expression in response to treatments

  • Evaluate purine supplementation efficacy using antibody-based detection of pathway components

• Patient-Derived Cell Studies:

  • Compare ADSL levels in cells from patients vs. healthy controls

  • Examine subcellular localization and potential aggregation in disease models

  • Correlate ADSL expression with neurological phenotype severity

What approaches can I use to study ADSL protein interactions using antibodies?

For investigating ADSL's protein-protein interactions:

• Co-Immunoprecipitation (Co-IP):

  • Use ADSL antibodies conjugated to agarose or magnetic beads

  • Identify interaction partners by Western blot or mass spectrometry

  • Confirm bidirectional interaction by reverse Co-IP

• Proximity Ligation Assay (PLA):

  • Detect protein interactions in situ with spatial resolution

  • Use antibodies against ADSL and potential interactors

  • Positive signal indicates proteins are within 40 nm proximity

• Immunofluorescence Co-localization:

  • Dual staining with ADSL and candidate interactor antibodies

  • Quantify co-localization using Pearson's or Mander's coefficients

  • Super-resolution microscopy for detailed interaction studies

• Bimolecular Fluorescence Complementation (BiFC):

  • Tag ADSL and potential interactor with split fluorescent protein fragments

  • Verify interactions in live cells

  • Validate with antibody detection of both proteins

• Crosslinking Immunoprecipitation (CLIP):

  • For studying ADSL-RNA interactions

  • Combine with high-throughput sequencing to identify binding sites

  • Validate with in vitro binding assays

How can ADSL antibodies be employed to investigate the relationship between ADSL deficiency and DNA damage?

ADSL deficiency has been linked to DNA damage and replication stress . To investigate this connection:

• DNA Damage Foci Analysis:

  • Co-stain ADSL-depleted cells with ADSL antibodies and DNA damage markers (53BP1, γH2AX)

  • Quantify foci number in ADSL-positive vs. ADSL-negative cells

  • Establish temporal relationship between ADSL depletion and damage appearance

• Cell Cycle Analysis:

  • Combine ADSL immunostaining with cell cycle markers (Ki67, BrdU incorporation)

  • Flow cytometry analysis using ADSL antibodies and propidium iodide

  • Correlate ADSL levels with G1 arrest phenotypes

• Chromatin Association Studies:

  • Perform chromatin fractionation followed by ADSL immunoblotting

  • Compare with RPA2 chromatin association (replication stress marker)

  • Determine if ADSL localizes to sites of DNA damage

• Rescue Experiments:

  • Test if nucleoside supplementation reduces DNA damage foci in ADSL-depleted cells

  • Compare effects of adenosine vs. complete nucleoside mixtures

  • Analyze pathway specificity using PAICS inhibitors vs. nucleoside supplementation

• p53 Dependency Analysis:

  • Co-stain for ADSL, p53, and DNA damage markers

  • Use p53 knockout/inhibition to determine dependency relationships

  • Monitor Ki67 levels to correlate with cell cycle exit

What quantitative methods are recommended for analyzing ADSL immunofluorescence data?

For robust quantitative analysis of ADSL immunostaining:

• Signal Intensity Measurement:

  • Measure mean fluorescence intensity within defined cellular compartments

  • Use software like ImageJ/FIJI with background subtraction

  • Compare intensity across experimental conditions using normalized values

• Co-localization Analysis:

  • Calculate Pearson's or Mander's coefficients for co-localization studies

  • Use JACoP or Coloc2 plugins in ImageJ for standardized analysis

  • Report thresholded coefficients to account for background

• Subcellular Distribution Profiles:

  • Generate fluorescence intensity profiles across cellular regions

  • Plot nuclear-to-cytoplasmic ratio to detect translocation events

  • Use mask-based approaches to quantify organelle-specific localization

• High-Content Analysis:

  • Automated image acquisition and analysis for large-scale studies

  • Multi-parametric phenotypic profiling

  • Machine learning approaches for pattern recognition and classification

• Statistical Considerations:

  • Use appropriate statistical tests (t-test, ANOVA, non-parametric tests)

  • Account for biological and technical replicates

  • Report effect sizes along with p-values

  • Consider blinded analysis to prevent bias

How do I reconcile contradictory results between different ADSL antibodies in my experiments?

When facing discrepancies between different ADSL antibodies:

• Epitope Mapping:

  • Identify the specific regions recognized by each antibody

  • Antibodies against different epitopes (e.g., AA 1-310 vs AA 188-237) may give different results if:

    • Certain epitopes are masked by protein interactions

    • Post-translational modifications affect epitope accessibility

    • Specific isoforms lack certain epitopes

• Validation Hierarchy:

  • Prioritize results from antibodies with more extensive validation

  • Consider evidence from knockout/knockdown controls

  • Weigh published literature support for each antibody

• Method-Specific Considerations:

  • Some antibodies work better for certain applications (WB vs. IF vs. IHC)

  • Native vs. denatured protein recognition can differ

  • Fixation methods may differentially affect epitope accessibility

• Orthogonal Approaches:

  • Use non-antibody methods to resolve discrepancies (mass spectrometry, CRISPR tagging)

  • Employ genetic approaches (tagged ADSL expression, CRISPR knockout)

  • Consider transcriptomics data to support protein findings

• Resolution Strategies:

  • Use multiple antibodies targeting different epitopes and report all results

  • Validate findings with genetic manipulations (siRNA, CRISPR)

  • Acknowledge limitations in your research publications

How can I distinguish between specific and non-specific effects when studying ADSL function using antibodies?

To ensure observed phenotypes are specifically related to ADSL function:

• Genetic Controls:

  • Use multiple siRNAs targeting different regions of ADSL mRNA

  • Perform rescue experiments with siRNA-resistant ADSL* constructs

  • Compare with CRISPR/Cas9-mediated ADSL knockout

• Pathway Validation:

  • Test if nucleoside supplementation rescues phenotypes

  • Inhibit upstream and downstream pathway components (e.g., PAICS inhibitor)

  • Correlate with metabolite measurements (purine levels)

• Cross-Species Validation:

  • Confirm findings across multiple model systems (human cells, zebrafish, chicken)

  • Use species-specific antibodies to verify knockdown efficiency

  • Compare phenotypes between models

• Dose-Response Relationships:

  • Establish quantitative relationships between ADSL levels and phenotype severity

  • Use partial knockdown approaches to reveal threshold effects

  • Correlate antibody signal intensity with functional outcomes

• Temporal Analysis:

  • Determine temporal sequence of events following ADSL depletion

  • Use inducible knockdown systems for temporal control

  • Establish which phenotypes appear first and may be causative rather than consequential

Antibody Product Information Table