ATX2 Antibody
Description
Definition and Basic Properties of ATX2 Antibody
The ATX2 antibody (Anti-ATXN2/Ataxin-2 Antibody) is a highly specific reagent designed to detect the ATXN2 protein, encoded by the ATXN2 gene. This protein contains polyglutamine tracts, and mutations in this gene are linked to neurodegenerative disorders such as spinocerebellar ataxia type 2 (SCA2), amyotrophic lateral sclerosis (ALS), and Parkinsonism . The antibody is validated for applications including:
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Western blot (WB)
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Immunohistochemistry (IHC)
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Immunofluorescence (IF)
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Flow Cytometry
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Immunocytochemistry (ICC)
Validation criteria:
Cytokinesis and Mitotic Regulation
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ATX-2 (ATXN2 homolog in C. elegans) regulates cytokinesis by modulating PAR-5 levels, ensuring proper localization of ZEN-4 (a kinesin) at spindle midzones .
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Depletion of ATX-2 causes cytokinesis defects, midzone microtubule disorganization, and cell cycle delays .
RNA Metabolism and Synaptic Plasticity
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ATX2 facilitates miRNA-mediated translational repression via interactions with Ago1 and Me31B, critical for long-term memory and synaptic plasticity .
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TRIBE analyses show ATX2 stabilizes target mRNAs (53.2% of targets) and destabilizes others (8.8%) in neuronal tissues .
Neurodegeneration and Cytoskeletal Dynamics
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Structured domains (e.g., LSm) protect against neurodegeneration, while disordered regions (IDRs) contribute to cytotoxicity .
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ATX2 regulates mRNA stability of cytoskeletal components, impacting neuronal trafficking and organelle dynamics .
Domain-Specific Functions
Mechanistic highlights:
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LSmAD lacks independent RNA-binding capacity but stabilizes LSm-RNA interactions .
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ATX2 antagonizes stress granule formation via LSm-PABP interplay, influencing translation under cellular stress .
Clinical and Therapeutic Implications
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
How should researchers validate ATX2 antibody specificity in cross-species models?
Validation requires a multi-modal approach:
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Western blot: Compare lysates from wild-type and ATX2-knockout models (e.g., C. elegans or human cell lines) to confirm band absence in knockout samples .
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Immunocytochemistry/Immunofluorescence (ICC/IF): Verify subcellular localization patterns against established ATX2 markers (e.g., cytoplasmic RNA granules or spindle midzone structures) .
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Cross-reactivity testing: Use phylogenetic alignment tools to assess epitope conservation between species. For example, C. elegans ATX-2 shares 43% identity with human ATXN2 in the N-terminal domain .
Table 1: Validation Techniques for ATX2 Antibodies
| Method | Key Parameters | Validation Criterion |
|---|---|---|
| Western Blot | Lysate source, denaturation conditions | Single band at predicted molecular weight |
| ICC/IF | Fixation method (e.g., paraformaldehyde) | Co-localization with known ATX2 markers |
| Knockout Verify | CRISPR/Cas9-modified models | Signal absence in null mutants |
What are the primary experimental applications of ATX2 antibodies in cell biology?
ATX2 antibodies enable three key applications:
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Cytokinesis studies: Monitor ATX2-PAR-5-ZEN-4 interactions during spindle midzone formation using time-lapse microscopy with GFP-tagged constructs .
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RNA granule dynamics: Quantify stress granule formation ratios under oxidative stress using high-content imaging (≥50 cells/condition) .
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Disease modeling: Detect polyQ-expanded ATXN2 aggregates in induced pluripotent stem cell (iPSC)-derived neurons via TR-FRET assays .
How to resolve contradictions in ATX2 localization data across studies?
Discrepancies often stem from:
Factor 1: Cell cycle phase dependency
ATX2 exhibits dynamic redistribution:
Factor 2: Antibody epitope accessibility
Comparative studies show:
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N-terminal antibodies detect 78% of total ATX2 in fixed cells vs. 34% in live imaging
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C-terminal antibodies fail to recognize phosphorylated isoforms (pSer625/628)
Critical validation step:
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Perform reciprocal immunoprecipitation with PAR-5 and ZEN-4 antibodies to confirm interaction hierarchy
Case Study: Resolving ATX2-PAR-5 Binding Dynamics
Contradiction: PAR-5 levels increase 1.55-fold in atx-2(ne4297) mutants , yet ZEN-4 localization requires ATX2-mediated PAR-5 suppression.
Resolution protocol:
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Time-resolved microscopy: Image PAR-5-GFP/ZEN-4-mCherry every 30 sec from anaphase onset
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Quantitative analysis:
Interpretation: ATX2 regulates PAR-5 through post-transcriptional mechanisms, creating a spatial gradient essential for ZEN-4 retention .
Emerging Techniques: TR-FRET for Soluble ATX2 Quantification
The TR-FRET assay (Time-Resolved Förster Resonance Energy Transfer) enables precise measurement of pathogenic ATX2 isoforms:
Table 2: TR-FRET Optimization Parameters
| Parameter | Optimal Condition | Impact on Signal/Noise |
|---|---|---|
| Antibody pair | 21776-1-AP + MW1 | 4.7-fold specificity |
| Lysate concentration | 1.5 mg/mL | Prevents matrix effects |
| Incubation time | 18 hr at 4°C | Maximizes epitope access |
Key application: Detected 23% reduction in soluble ATX2 upon starvation (p=0.0082) in neuronal cultures .
How to analyze conflicting ATX2 functional data between model organisms?
Scenario: C. elegans studies emphasize cytokinesis roles , while Drosophila models show mRNA regulation .
Resolution framework:
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Orthology mapping:
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C. elegans ATX-2: 3 LSM domains, 1 PAM2 motif
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Human ATXN2: Expanded polyQ tract (+22 residues) alters interactome
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Conditional knockdown: Tissue-specific RNAi reveals 71% functional conservation in mitotic vs. 39% in neuronal contexts
Recommendation: Use cross-species antibody panels (see Table 1) to distinguish conserved vs. species-specific functions.
