ATXN2 Antibody

What epitopes do commercial ATXN2 antibodies typically target?

Commercial ATXN2 antibodies generally target either N-terminal or C-terminal regions of the protein. C-terminal antibodies (often referred to as C-ATXN2) are most common, with several validated options available across multiple suppliers . N-terminal antibodies are less common, and researchers often use epitope-tagging approaches (such as HA-tagging) when studying N-terminal fragments .

Some key epitope regions include:

• N-terminal regions upstream of the polyQ domain

• The polyQ domain itself (specifically targeted by antibodies like 1C2 that detect expanded polyQ tracts)

• Conserved domains such as the Lsm or LsmAD domains

• C-terminal regions containing the PAM2 domain

When selecting an antibody, consider which region of ATXN2 is most relevant to your research question and whether you need to distinguish between different isoforms or cleavage products .

How can I validate the specificity of an ATXN2 antibody?

Validating ATXN2 antibody specificity requires multiple complementary approaches:

• RNA interference validation: Create cell lines expressing different shRNAs targeting ATXN2 mRNA. A specific antibody should show reduced signal intensity in Western blots of these knockdown lines compared to controls, while maintaining consistent detection of control proteins like GAPDH .

• Overexpression testing: Express tagged versions of ATXN2 (e.g., HA-tagged ATXN2) and confirm co-detection with both the tag-specific antibody and your ATXN2 antibody .

• Immunoprecipitation: Perform IP with one antibody and detection with another targeting a different epitope to confirm specificity .

• Comparative analysis with multiple antibodies: Use multiple antibodies targeting different ATXN2 epitopes to verify consistent protein detection patterns .

• Negative controls: Include tissues or cell lines from ATXN2 knockout models when available .

Which applications are ATXN2 antibodies validated for?

Most commercial ATXN2 antibodies have been validated for multiple applications:

Multiple studies confirm Western blot and immunohistochemistry as the most reliable applications, with most antibodies detecting the expected ~145 kDa and ~180 kDa bands corresponding to different ATXN2 isoforms or cleavage products .

How can I detect polyQ-expanded ATXN2 versus normal ATXN2?

Detecting polyQ-expanded ATXN2 requires specialized approaches:

• PolyQ-specific antibodies: The 1C2 antibody (clone 5TF1-1C2) specifically recognizes expanded polyQ tracts (typically >37Q) but not normal-length polyQ domains. This allows selective detection of mutant ATXN2 .

• TR-FRET immunoassay: This highly sensitive approach uses two antibodies - one ATXN2-specific antibody labeled with a donor fluorophore (Tb) and one polyQ-specific antibody labeled with an acceptor fluorophore (D2). Energy transfer only occurs when both antibodies bind in close proximity, allowing selective detection of polyQ-expanded ATXN2 .

• Gel mobility shift analysis: PolyQ-expanded ATXN2 migrates more slowly in SDS-PAGE compared to normal ATXN2, creating a detectable mobility shift that correlates with polyQ length. This approach requires high-resolution gels and optimized running conditions .

• Combination approach: For highest specificity, combine a general ATXN2 antibody with a polyQ-specific antibody (like 1C2) on parallel blots of the same samples .

The TR-FRET method offers particular advantages for quantitative analysis, with sensitivity sufficient to detect small changes in protein expression following treatments like siRNA knockdown or starvation .

How can I establish a TR-FRET-based immunoassay for quantifying polyQ-expanded ATXN2?

Establishing a TR-FRET immunoassay for polyQ-expanded ATXN2 requires several optimization steps:

• Antibody selection and labeling:

  • Select two ATXN2-specific antibodies (e.g., ataxin-2 polyclonal antibody 21776-1-AP from Proteintech and purified mouse anti-ataxin-2 monoclonal antibody AB_398900 from BD)

  • Label them with donor fluorophore Tb

  • Select polyQ-specific antibodies (e.g., clone MW1 AB_528290 or clone 5TF1-1C2 MAB1574)

  • Label them with acceptor fluorophore D2

• Buffer optimization:

  • Test different buffer conditions to maximize signal-to-background ratio

  • Include protease inhibitors to prevent protein degradation

  • Optimize detergent concentrations to maintain protein solubility while preserving antibody binding

• Assay validation:

  • Use recombinant ATXN2 with normal (22Q) and expanded (79Q) polyQ tracts as controls

  • Validate in cellular models (e.g., HEK293T cells transfected with ATXN2 constructs)

  • Test in patient-derived materials like iPSC-neurons

• Sensitivity testing:

  • Determine lower limits of detection

  • Validate ability to detect changes following treatments (e.g., siRNA knockdown)

The established TR-FRET method has demonstrated sufficient sensitivity to detect small changes in ATXN2 expression and can be applied to various biological samples including cell lysates and potentially biofluids .

What techniques can detect ATXN2 N-terminal proteolytic fragments?

Detecting ATXN2 N-terminal fragments requires specialized techniques:

Remember that N-terminal ATXN2 fragments containing expanded polyQ tracts have been observed in brain extracts from SCA2 patients and may have pathological significance .

How can I analyze ATXN2 subcellular localization in neuronal models?

Analyzing ATXN2 subcellular localization requires careful experimental design:

• Co-localization studies:

  • Combine ATXN2 antibodies with markers for specific subcellular compartments

  • Ribosomal protein S6 and poly-A binding protein 1 (translation machinery)

  • Calnexin (endoplasmic reticulum)

  • TIA-1 or G3BP1 (stress granules)

  • pTDP-43 (in disease models)

• Fixation optimization:

  • Different fixation methods preserve different protein interactions

  • Paraformaldehyde fixation (4%, 10-15 minutes) works well for most applications

  • For stress granule co-localization, brief methanol fixation may better preserve RNA-protein complexes

• Super-resolution microscopy:

  • Standard confocal microscopy may not resolve fine structures

  • STED or STORM microscopy provides enhanced resolution to distinguish ATXN2 within complex structures like stress granules

• Live-cell imaging:

  • For dynamic processes like stress granule formation, use fluorescently-tagged ATXN2 constructs

  • Verify that tagging doesn't alter localization by comparing with immunostaining of endogenous ATXN2

• Biochemical fractionation:

  • Complement imaging with subcellular fractionation (cytosolic, membrane, nuclear fractions)

  • Use Western blotting with ATXN2 antibodies to quantify distribution

Normal ATXN2 typically localizes to the cytoplasm and proximal dendrites in neurons, with strong association with protein synthesis machinery, while expanded ATXN2 may show altered localization patterns in disease models .

What are the best approaches for investigating ATXN2 in post-mortem human brain samples?

Working with post-mortem tissue requires specialized approaches:

• Sample preparation:

  • Optimize tissue preservation - fresh frozen tissue generally yields better results than formalin-fixed paraffin-embedded tissue

  • For protein extraction, use buffers containing detergents like sarkosyl to solubilize membrane-associated proteins

  • Separate sarkosyl-soluble and sarkosyl-insoluble fractions to distinguish aggregated from soluble ATXN2

• Immunohistochemistry protocol:

  • Use heat-mediated antigen retrieval (citrate buffer pH 6.0)

  • Block endogenous peroxidases and biotin

  • Include Sudan Black B treatment to reduce autofluorescence if using fluorescent detection

  • Use Tyramide Signal Amplification for weak signals

• Regional analysis:

  • Focus on cerebellar Purkinje cells (primary pathology in SCA2)

  • Also examine hippocampal and cortical regions (relevant for TDP-43 pathology)

• Co-localization studies:

  • In FTLD-TDP cases, examine co-localization with phosphorylated TDP-43 in neuronal cytoplasmic inclusions and dystrophic neurites

  • Use sequential double-labeling techniques to minimize cross-reactivity

• Quantitative analysis:

  • Use semi-quantitative immunofluorescence analysis to measure ATXN2 levels

  • Compare protein levels between disease cases and controls using Western blot densitometry

Studies of FTLD-TDP cases have revealed colocalization of ATXN2 with phosphorylated TDP-43 in pathological inclusions and significant reduction of ATXN2 protein compared to controls, suggesting involvement in TDP-43 proteinopathies .

How do I distinguish between different ATXN2 isoforms and cleavage products?

Distinguishing between ATXN2 isoforms requires multiple analytical approaches:

• Molecular weight analysis:

  • Full-length ATXN2 (from ATG1 start site): ~180 kDa apparent molecular weight

  • Shorter ATXN2 isoform (from ATG2 start site): ~145 kDa

  • N-terminal cleavage products with normal polyQ: ~27 kDa

  • N-terminal cleavage products with expanded polyQ: ~30 kDa

  • C-terminal fragments lacking polyQ domain: ~145 kDa or smaller

• Epitope mapping:

  • Use N-terminal tag antibodies (e.g., HA) to detect N-terminal fragments

  • Use C-terminal ATXN2 antibodies to detect C-terminal fragments

  • Use polyQ-specific antibodies (1C2) to confirm polyQ presence in fragments

• Expression constructs:

  • Express ATXN2 from specific start codons (ATG1 vs. ATG2)

  • Create deletion constructs to identify cleavage sites

  • Compare migration patterns on Western blots

• Immunoprecipitation:

  • IP with N-terminal antibodies to determine which fragments remain associated after cleavage

  • IP with C-terminal antibodies to pull down C-terminal fragments

• Gradient gel electrophoresis:

  • Use 4-20% gradient gels to resolve both high and low molecular weight species

  • Apply appropriate molecular weight markers spanning the full range

Research has shown that full-length ATXN2 migrates more slowly than predicted (~180 kDa vs. predicted ~140 kDa), while N-terminal cleavage products containing the polyQ domain show mobility differences that correlate with polyQ length .

What are the critical factors in developing PolyQ-specific detection of ATXN2?

Developing polyQ-specific detection systems requires attention to several critical factors:

• Antibody selection:

  • The monoclonal antibody 1C2 (clone 5TF1-1C2, MAB1574) specifically recognizes expanded polyQ stretches (typically >37Q)

  • Alternative polyQ antibodies include clone MW1 (AB_528290)

  • Different polyQ antibodies may have different threshold sensitivities for polyQ length

• Signal-to-noise optimization:

  • Include appropriate negative controls (cells expressing normal-length ATXN2)

  • Titrate antibody concentrations to minimize background

  • For TR-FRET assays, optimize donor/acceptor antibody ratios

• Buffer conditions:

  • Detergent selection affects polyQ epitope accessibility

  • Salt concentration can influence antibody specificity

  • pH optimization may be necessary for maximum binding

• PolyQ length considerations:

  • Establish detection thresholds for different polyQ lengths

  • Use constructs with varying polyQ lengths (e.g., 22Q, 39Q, 79Q) as standards

  • Account for potential CAA interruptions in the CAG repeat that may affect antibody binding

• Protein conformation:

  • Native vs. denatured protein may affect polyQ accessibility

  • Include protein denaturants like SDS for Western blot applications

  • Consider native conditions for TR-FRET applications

TR-FRET-based methods combining ATXN2-specific and polyQ-specific antibodies offer particular advantages for sensitive and specific detection of expanded ATXN2, with demonstrated utility in monitoring treatment effects in cellular models .

How can I monitor changes in ATXN2 levels following experimental treatments?

Monitoring ATXN2 changes requires quantitative approaches:

• Western blot quantification:

  • Use housekeeping proteins (β-actin, GAPDH) as loading controls

  • Apply densitometry analysis with appropriate software

  • Run standard curves using recombinant ATXN2 for absolute quantification

• TR-FRET quantification:

  • Particularly useful for detecting small changes in polyQ-expanded ATXN2

  • Has been validated for monitoring siRNA knockdown effects

  • Can detect changes following cellular stresses like starvation

• qPCR for transcript changes:

  • Design primers spanning exon-exon junctions

  • Include reference genes (GAPDH, ACTB)

  • Correlate with protein changes to identify post-transcriptional regulation

• Immunocytochemistry with quantitative image analysis:

  • Use consistent acquisition parameters

  • Apply automated image analysis for unbiased quantification

  • Include nuclear or cytoskeletal markers for normalization

• Experimental timeline considerations:

  • For siRNA experiments, 72-hour post-transfection timepoint has been validated

  • For starvation experiments, effects have been observed within 1-2 hours

The TR-FRET immunoassay has been validated as sufficiently sensitive to detect small changes in ATXN2 expression following treatments like siRNA knockdown or starvation, making it particularly valuable for therapeutic monitoring applications .

What are the key considerations when studying ATXN2 and TDP-43 interactions?

Studying ATXN2-TDP-43 interactions requires specialized approaches:

• Co-immunoprecipitation strategy:

  • Use antibodies against native proteins or epitope tags

  • Include RNase treatment controls to distinguish RNA-dependent interactions

  • Use crosslinking approaches for transient interactions

• Co-localization analysis:

  • In FTLD-TDP cases, focus on hippocampal regions

  • Look for colocalization in both neuronal cytoplasmic inclusions and dystrophic neurites

  • Use phospho-TDP-43 specific antibodies to identify pathological TDP-43

• Genetic modification approaches:

  • Use ATXN2 knockdown to assess effects on TDP-43 aggregation

  • Compare normal vs. expanded polyQ ATXN2 effects on TDP-43 localization

  • Consider intermediate polyQ expansions relevant to ALS/FTLD

• Stress response analysis:

  • Examine stress granule dynamics where both proteins localize

  • Apply stressors like arsenite, heat shock, or starvation

  • Monitor recovery phases to assess granule persistence

• Biochemical fractionation:

  • Separate detergent-soluble and insoluble fractions

  • Compare distribution of both proteins across fractions

  • Assess changes in solubility following stress or in disease models

Research has shown reduced ATXN2 protein levels in FTLD-TDP cases compared to controls, with ATXN2 colocalizing with phosphorylated TDP-43 in pathological inclusions, suggesting a functional relationship relevant to disease mechanisms .

What controls are essential when studying ATXN2 proteolytic cleavage?

Studying ATXN2 proteolytic cleavage requires rigorous controls:

• Construct controls:

  • Compare full-length (ATG1) vs. shortened (ATG2) ATXN2 constructs

  • Include both normal (22Q) and expanded (39Q/79Q) polyQ variants

  • Generate deletion constructs to map cleavage sites

• Protease inhibitor controls:

  • Include protease inhibitor cocktails in some samples

  • Test specific inhibitors to identify protease classes involved

  • Compare fresh vs. delayed extraction to assess post-lysis artifacts

• Fragment authentication:

  • Confirm N-terminal fragments using both tag antibodies and polyQ antibodies

  • Verify C-terminal fragments using C-terminal ATXN2 antibodies

  • Perform immunoprecipitation to confirm fragment identity

• Heterologous protein controls:

  • Test putative cleavage sequences by inserting into heterologous proteins

  • Confirm sufficiency of identified sequences for directing proteolysis

  • Use irrelevant sequences as negative controls

• In vivo relevance controls:

  • Compare cellular findings with patient tissue samples

  • Analyze both control and disease brain extracts

  • Consider post-mortem interval effects on proteolysis

Research has demonstrated that both normal and expanded polyQ ATXN2 undergo proteolytic cleavage, releasing polyQ-containing N-terminal fragments, with potential implications for SCA2 pathogenesis similar to other polyQ disorders .

How can ATXN2 antibodies be used to evaluate therapeutic efficacy in SCA2 models?

ATXN2 antibodies offer several approaches for therapeutic monitoring:

• Protein lowering assessment:

  • Quantify ATXN2 reduction following ASO or siRNA treatments

  • Use TR-FRET assays for sensitive detection of polyQ-expanded ATXN2

  • Measure both mRNA and protein levels to assess correlation

• Tissue-specific evaluation:

  • Assess treatment effects across different tissues (cerebellum, brainstem, spinal cord)

  • Use immunohistochemistry to evaluate regional differences in protein lowering

  • Compare effects in neurons vs. glial cells

• Isoform-specific analysis:

  • Determine whether treatments affect all ATXN2 isoforms equally

  • Monitor both full-length protein and proteolytic fragments

  • Assess normal vs. expanded polyQ ATXN2 selectivity

• Cellular phenotype correlation:

  • Relate ATXN2 reduction to cellular phenotypes (stress granule dynamics, translation efficiency)

  • Correlate with downstream pathway markers

  • Use proximity ligation assays to assess protein-protein interactions

• Biofluid biomarker development:

  • Apply TR-FRET assays to CSF or plasma samples

  • Establish baseline measures and variation in patient cohorts

  • Track longitudinal changes during therapeutic interventions

The established TR-FRET immunoassay for polyQ-expanded ATXN2 has demonstrated sufficient sensitivity for therapeutic monitoring applications and has potential utility as a pharmacodynamic biomarker in clinical trials .

What are the challenges in developing ATXN2 as a biomarker for neurodegenerative diseases?

Developing ATXN2 as a biomarker faces several challenges:

• Sample accessibility:

  • ATXN2 levels in accessible biofluids (blood, CSF) may not reflect brain levels

  • Optimization of extraction protocols for different sample types is needed

  • Blood-brain barrier limits CNS protein detection in peripheral samples

• Assay sensitivity requirements:

  • Low abundance in biofluids requires highly sensitive detection methods

  • TR-FRET approaches offer promising sensitivity but need optimization for biofluids

  • Amplification methods may be necessary for reliable detection

• Isoform complexity:

  • Multiple ATXN2 isoforms and cleavage products complicate interpretation

  • Need for assays that distinguish pathogenic from normal forms

  • Potential post-translational modifications affecting detection

• Disease specificity considerations:

  • ATXN2 alterations occur in multiple disorders (SCA2, ALS, FTLD-TDP)

  • Need to establish disease-specific patterns of change

  • Requirement for differential diagnostic potential

• Validation requirements:

  • Correlation with clinical measures and disease progression

  • Establishment of age and sex-specific reference ranges

  • Validation across multiple cohorts and centers

The development of TR-FRET-based immunoassays specific for polyQ-expanded ATXN2 represents a promising approach for biomarker development, with demonstrated sensitivity in cellular models, but additional validation in patient biofluids is still needed .