ATXN1L Antibody

What is ATXN1L and why is it important in research?

ATXN1L (Ataxin-1-Like) is a paralog of ATXN1 that functions as a chromatin-binding factor involved in transcriptional repression. It specifically represses Notch signaling by acting as a CBF1 corepressor and binds to the HEY promoter, potentially assisting RBPJ-mediated repression alongside NCOR2 . ATXN1L has gained significant research interest because it can functionally compensate for ATXN1, whose polyglutamine expansion causes spinocerebellar ataxia type 1 (SCA1). Molecular studies have shown that mild overexpression of ATXN1L can suppress several pathological phenotypes associated with both ATXN1 loss-of-function and polyglutamine-expanded ATXN1, making it a potential therapeutic target for SCA1 . Additionally, recent research has uncovered an immunomodulatory role for ATXN1 and by extension ATXN1L, particularly in regulating B cell function and T helper type 1 (Th1) differentiation, further broadening its research significance .

What are the key characteristics of commercially available ATXN1L antibodies?

Commercial ATXN1L antibodies are available in several formats, with the most common being rabbit polyclonal antibodies. For instance, Abcepta offers an affinity-purified rabbit polyclonal antibody targeting the central region (amino acids 385-411) of human ATXN1L . Similarly, Sigma-Aldrich produces a rabbit polyclonal antibody (HPA062789) that targets the sequence "HPGIHYPPLHYAQLPSTSLQFIGSPYSLPYAVPPNFLPSPLLSPSANLATSHLPHFVPYASLLAEGATPP" of human ATXN1L . These antibodies are typically supplied in buffered solutions with preservatives like sodium azide and are designed for applications including Western blotting and immunofluorescence. The recommended dilution for Western blotting is typically 1:1000 , while for immunofluorescence, concentration ranges of 0.25-2 μg/mL may be used . The antibodies show primary reactivity to human ATXN1L with predicted cross-reactivity to mouse ATXN1L due to sequence conservation .

How does ATXN1L differ functionally from ATXN1?

The most significant functional difference between these proteins emerges in pathological contexts: while mutant forms of ATXN1 with expanded polyglutamine tracts cause SCA1 through a toxic gain-of-function mechanism, ATXN1L does not undergo similar pathogenic expansion. Instead, ATXN1L can ameliorate SCA1 pathogenesis by two distinct mechanisms: (1) enhancing the formation of ATXN1L-Cic complexes, thereby stabilizing Cic protein levels that are reduced in Atxn1-/- mice, and (2) inducing the sequestration of polyglutamine-expanded ATXN1 into nuclear inclusions, potentially displacing the mutant protein from its endogenous complexes . These functional differences highlight ATXN1L's potential therapeutic role in SCA1 and related neurodegenerative disorders.

How do experimental conditions affect ATXN1L antibody specificity and sensitivity?

The specificity and sensitivity of ATXN1L antibodies can be significantly affected by several experimental conditions that researchers must carefully control. Fixation methods can dramatically impact epitope accessibility, particularly for nuclear proteins like ATXN1L. Paraformaldehyde fixation typically preserves ATXN1L epitopes better than methanol-based protocols, though this can vary based on the specific antibody and targeted epitope region.

The blocking agent selection is another critical factor. When using ATXN1L antibodies for immunoblotting, BSA sometimes yields lower background compared to milk-based blockers, as milk proteins may contain phospho-epitopes that could cross-react with some antibodies. For immunoprecipitation experiments with ATXN1L, as demonstrated in studies of ATXN1L-Cic complexes, buffer composition significantly affects complex stability. The interaction studies showing increased ATXN1L-Cic co-immunoprecipitation in Atxn1-/- cerebella utilized optimized buffer conditions that maintained these protein complexes .

Temperature and incubation time also affect antibody performance. For the Abcepta ATXN1L antibody with a recommended 1:1000 dilution for Western blotting , overnight incubation at 4°C generally provides better signal-to-noise ratio than shorter incubations at room temperature, particularly when detecting endogenous levels of ATXN1L in complex tissue samples like cerebellum.

What are the challenges in detecting endogenous ATXN1L in different tissue and cell types?

Detecting endogenous ATXN1L presents several tissue-specific challenges that researchers must address through methodological refinements. In neuronal tissues, particularly cerebellum where ATXN1L function has been extensively studied in relation to SCA1 pathogenesis , the high lipid content can interfere with protein extraction and subsequent detection. Enhanced lysis buffers containing 1-2% SDS or specialized lipid-removal steps prior to immunoblotting can improve detection.

ATXN1L detection in immune cells presents different challenges. In B cells, where ATXN1 (and potentially ATXN1L) regulates key costimulatory molecules like CD44 and CD80 , high protein turnover rates can result in variable detection levels. Proteasome inhibitors like MG132 added shortly before cell harvesting can stabilize ATXN1L for more consistent detection.

Another significant challenge is distinguishing ATXN1L from ATXN1 due to their sequence homology. Strategic antibody selection targeting unique regions is crucial. The Abcepta antibody targeting amino acids 385-411 and Sigma-Aldrich's HPA062789 both target sequences with minimal overlap to ATXN1, reducing cross-reactivity.

The subcellular localization of ATXN1L also impacts detection sensitivity. As a nuclear protein involved in chromatin binding, nuclear extraction protocols yield better results than total protein extraction methods. Using stepwise extraction protocols that separate cytoplasmic and nuclear fractions can significantly improve detection sensitivity when using techniques like immunofluorescence or immunohistochemistry.

How can contradictory experimental results with ATXN1L antibodies be reconciled?

Contradictory results when using ATXN1L antibodies often stem from several factors that can be systematically addressed through experimental design refinements. First, epitope availability varies between experimental systems. ATXN1L forms complexes with proteins like Cic , which may mask antibody epitopes depending on the experimental context. Performing parallel experiments with antibodies targeting different ATXN1L regions can help verify results. For instance, comparing results from antibodies targeting the C-terminal region versus the central domain (amino acids 385-411) can reveal context-dependent protein interactions.

Post-translational modifications also contribute to contradictory findings. The functional state of ATXN1L may involve phosphorylation or other modifications that alter antibody recognition. Western blot analysis often reveals multiple bands representing different modified forms of ATXN1L. Treatment with phosphatases or other modification-removing enzymes before immunodetection can clarify whether modifications contribute to contradictory results.

A particularly challenging scenario arises when comparing ATXN1L detection between wildtype and disease models. For example, in SCA1 models, where ATXN1L overexpression suppresses phenotypes , altered protein complex formation may change epitope accessibility. In these cases, employing multiple detection methods (immunoprecipitation followed by mass spectrometry alongside standard immunoblotting) can provide complementary data to resolve contradictions.

What are the optimal protocols for immunoprecipitation of ATXN1L-containing protein complexes?

Immunoprecipitation of ATXN1L-containing complexes requires careful optimization to maintain physiologically relevant interactions while achieving sufficient yield. Based on successful studies of ATXN1L-Cic interactions , the following protocol has proven effective:

Optimized ATXN1L Complex Immunoprecipitation Protocol:

• Tissue/Cell Preparation:

  • For tissue samples (e.g., cerebellum), homogenize in ice-cold IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM EDTA) supplemented with protease inhibitors and phosphatase inhibitors.

  • For cell culture, harvest cells in the same buffer after washing with cold PBS.

• Lysis Conditions:

  • Include 10% glycerol in the lysis buffer to stabilize protein complexes during extraction.

  • Incubate lysates on ice for 30 minutes with gentle agitation every 5-10 minutes.

  • Centrifuge at 14,000 × g for 15 minutes at 4°C to remove insoluble material.

• Pre-clearing Step:

  • Pre-clear lysates with Protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

  • This step is particularly important when working with tissue samples that contain high levels of endogenous immunoglobulins.

• Antibody Incubation:

  • For ATXN1L immunoprecipitation, use 2-5 μg of ATXN1L antibody per 1 mg of protein lysate.

  • For Cic immunoprecipitation to pull down ATXN1L-Cic complexes, use Cic-specific antibodies following similar ratios.

  • Incubate overnight at 4°C with gentle rotation.

• Bead Capture and Washing:

  • Add pre-equilibrated Protein A/G beads and incubate for 2-3 hours at 4°C.

  • Perform 4-5 washes with washing buffer (IP buffer with reduced NP-40 to 0.1%).

  • Include a final wash with detergent-free buffer to remove residual detergent.

• Elution and Analysis:

  • Elute complexes with 2X SDS sample buffer at 70°C rather than boiling to preserve complex integrity.

  • Analyze by Western blotting using 1:1000 dilution of ATXN1L antibody or antibodies against interacting partners.

This protocol has successfully demonstrated increased ATXN1L-Cic co-immunoprecipitation in Atxn1-/- cerebella compared to wild-type samples, supporting its efficacy for studying physiologically relevant ATXN1L complexes .

How should Western blotting protocols be optimized for reliable ATXN1L detection?

Reliable detection of ATXN1L by Western blotting requires specific optimization strategies due to its nuclear localization and involvement in protein complexes. The following protocol incorporates modifications proven effective for detection of ATXN1L with its calculated molecular weight of approximately 73 kDa :

Optimized Western Blotting Protocol for ATXN1L:

• Sample Preparation:

  • For nuclear proteins like ATXN1L, include a nuclear extraction step using a high-salt buffer (300-400 mM NaCl) to ensure efficient extraction.

  • Add denaturation buffer containing 2% SDS and 5% β-mercaptoethanol.

  • Heat samples at 70°C for 10 minutes rather than boiling to prevent protein aggregation.

• Gel Electrophoresis:

  • Use gradient gels (4-12% or 4-15%) to achieve optimal separation around the 73 kDa region.

  • Include molecular weight markers that clearly mark the 70-80 kDa range.

  • Load 30-40 μg of total protein for cell lysates or 20-25 μg for nuclear extracts.

• Transfer Conditions:

  • Transfer to PVDF membranes (0.45 μm pore size) at 30V overnight at 4°C.

  • Include 10-20% methanol in transfer buffer to enhance protein binding to the membrane.

  • Verify transfer efficiency using reversible protein stains before blocking.

• Blocking and Antibody Incubation:

  • Block with 5% BSA in TBST for 1-2 hours at room temperature.

  • Incubate with primary ATXN1L antibody at 1:1000 dilution in TBST with 1% BSA overnight at 4°C.

  • Wash extensively (5 × 5 minutes) with TBST.

  • Incubate with HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour at room temperature.

• Signal Development:

  • Use enhanced chemiluminescence (ECL) with extended exposure times (2-5 minutes).

  • For low-abundance detection, consider using signal enhancers or more sensitive ECL substrates.

• Controls and Validation:

  • Include positive controls from tissues known to express ATXN1L (e.g., cerebellum).

  • Use Atxn1L-/- tissues or knockdown cell lysates as negative controls when available.

  • Consider checking for cross-reactivity with recombinant ATXN1 protein to ensure specificity.

This optimized protocol addresses the challenges specific to ATXN1L detection and has been shown to reliably detect both endogenous ATXN1L and overexpressed ATXN1L in various experimental contexts .

What are the best approaches for measuring ATXN1L-mediated transcriptional repression?

ATXN1L functions as a transcriptional repressor, particularly in Notch signaling pathways . Measuring its repressive activity requires specialized approaches that assess its impact on target gene expression. The following methodologies have proven effective:

Luciferase Reporter Assays:

• Design reporter constructs containing ATXN1L-responsive elements, particularly those from the HEY promoter or other known targets .

• Co-transfect cells with the reporter construct, a constitutively active luciferase control, and either ATXN1L expression vectors or siRNAs.

• After 24-48 hours, measure luciferase activity and normalize to the constitutive control.

• Compare repression efficiency between wild-type ATXN1L and mutant variants or in the presence of interacting proteins like Cic.

Chromatin Immunoprecipitation (ChIP):

• Cross-link protein-DNA complexes in cells expressing endogenous or tagged ATXN1L.

• Immunoprecipitate chromatin using ATXN1L antibodies (1-2 μg/reaction) .

• Analyze enrichment at target genomic regions using qPCR or sequencing.

• Include controls for non-specific binding and validate with known ATXN1L targets.

Gene Expression Analysis:

• Manipulate ATXN1L levels through overexpression or knockdown in relevant cell types.

• Extract RNA and perform RT-qPCR to measure changes in known target genes.

• For genome-wide effects, use RNA-seq to identify differentially expressed genes.

• Validate direct targets through motif analysis and comparison with ChIP data.

Research has shown that ATXN1L, similar to ATXN1, regulates gene expression by forming complexes with Cic. The comparative analysis of gene expression in Atxn1-/- mice with and without ATXN1L overexpression has been particularly informative, revealing that ATXN1L can functionally compensate for ATXN1 in transcriptional regulation . This approach effectively differentiates between direct ATXN1L targets and secondary effects, providing a comprehensive view of ATXN1L's role in transcriptional repression.

How should researchers analyze ATXN1L expression across different tissues and disease states?

Analyzing ATXN1L expression across tissues and disease states requires a systematic approach that accounts for tissue-specific variations and disease-related changes. Based on research practices that have yielded valuable insights into ATXN1L function, the following analytical framework is recommended:

Tissue Expression Profiling:

• Establish baseline expression using multiple detection methods (qRT-PCR, Western blotting, and immunohistochemistry) to create a comprehensive expression atlas.

• When comparing expression levels across tissues, use tissue-specific housekeeping genes for normalization rather than global standards.

• Account for potential splicing isoforms of ATXN1L that may be differentially expressed across tissues, as observed with ATXN1 in splenocytes during experimental autoimmune encephalomyelitis (EAE) peak .

Disease State Analysis:

• Compare ATXN1L expression between matched healthy and diseased tissues, controlling for age, sex, and genetic background.

• Consider temporal dynamics of expression, especially in progressive disorders like SCA1, where ATXN1L's compensatory effects may vary with disease stage .

• Analyze not only absolute expression levels but also the relative distribution between different protein complexes, as ATXN1L's function depends significantly on its interaction partners like Cic .

Data Integration Framework:

This multi-level analysis approach has revealed important insights, such as the increased formation of ATXN1L-Cic complexes in Atxn1-/- cerebella despite reduced Cic protein levels, suggesting compensatory mechanisms that can be therapeutically relevant .

What analytical approaches help differentiate between ATXN1L and ATXN1 functions in experimental models?

Differentiating between ATXN1L and ATXN1 functions requires sophisticated analytical approaches that can untangle their overlapping yet distinct roles. Based on successful experimental paradigms, the following analytical strategies are recommended:

Genetic Complementation Analysis:

• Use rescue experiments in which ATXN1L is expressed in Atxn1-/- backgrounds to identify which phenotypes can be complemented by ATXN1L.

• Quantify the degree of rescue for different phenotypes, establishing a hierarchy of ATXN1-specific versus shared functions.

• This approach has successfully demonstrated that mild overexpression of ATXN1L can rescue several molecular and behavioral defects in Atxn1-/- mice, indicating functional overlap .

Interactome Differential Analysis:

• Compare protein interaction profiles of ATXN1L and ATXN1 using affinity purification-mass spectrometry.

• Focus on unique and shared interactors, particularly the relative affinity for common partners like Cic.

• Analyze how these interaction patterns change in disease contexts or upon genetic manipulation.

Target Gene Specificity Mapping:

• Perform ChIP-seq for both ATXN1L and ATXN1 to identify genome-wide binding sites.

• Use differential binding analysis to categorize targets as ATXN1-specific, ATXN1L-specific, or shared.

• Correlate binding patterns with gene expression changes in single and double knockout models.

Domain Swap Experiments:

• Create chimeric proteins containing domains from both ATXN1 and ATXN1L.

• Test these chimeras in functional assays to map domain-specific activities.

• Combine with structural biology approaches to understand the molecular basis of functional differences.

These analytical approaches have revealed several key distinctions: while both proteins form complexes with Cic, ATXN1L appears to stabilize these complexes more effectively when ATXN1 is absent. Additionally, ATXN1L overexpression can prevent the destabilization of Cic that occurs in Atxn1-/- cerebellum , providing evidence for both redundant and specific functions. These findings support a model in which ATXN1L can functionally compensate for ATXN1 in certain contexts, particularly in transcriptional regulation through Cic-containing complexes.

How can researchers interpret contradictory findings regarding ATXN1L's role in disease pathogenesis?

Interpreting contradictory findings about ATXN1L's role in disease pathogenesis requires a systematic framework that considers context-dependent factors and methodological differences. The following analytical strategies help reconcile apparently conflicting results:

Context-Dependency Analysis:

• Stratify findings based on experimental context (in vitro versus in vivo, cell type, disease model).

• Map contradictory results against biological variables like developmental stage, disease progression, and genetic background.

• This approach has helped reconcile findings about ATXN1L in SCA1, where its protective effects vary depending on the disease stage and cellular context .

Methodological Reconciliation Framework:

• Categorize contradictions based on whether they arise from different methodologies, model systems, or interpretation frameworks.

• Perform bridging experiments that replicate key findings using standardized protocols.

• For antibody-related contradictions, validate with multiple antibodies targeting different epitopes .

Dosage-Effect Analysis:

• Systematically analyze how different expression levels of ATXN1L influence experimental outcomes.

• Create dose-response curves for phenotypic rescue or exacerbation in disease models.

• This has been critical in understanding ATXN1L's role in SCA1, where mild overexpression is protective, potentially by enhancing the formation of ATXN1L-Cic complexes .

Molecular Mechanism Integration:

• Develop integrated models that incorporate seemingly contradictory findings into a coherent mechanism.

• Test these models with targeted experiments that differentiate between alternative explanations.

A prime example of successful reconciliation concerns ATXN1L's role in SCA1 pathogenesis. Initially, seemingly contradictory findings suggested both protective and neutral roles for ATXN1L. These were reconciled through the discovery of a dual mechanism: ATXN1L can both compensate for partial loss of ATXN1 function by stabilizing Cic-containing complexes and simultaneously suppress toxicity by sequestering polyglutamine-expanded ATXN1 . This integrated model explains why ATXN1L overexpression is protective in SCA1 models and provides a framework for therapeutic development.

What are emerging applications of ATXN1L antibodies in neurodegenerative disease research?

ATXN1L antibodies are finding increasingly sophisticated applications in neurodegenerative disease research, with several emerging directions showing particular promise:

Biomarker Development:

• ATXN1L antibodies are being explored for developing assays to measure ATXN1L/ATXN1 ratios in cerebrospinal fluid and blood, potentially providing prognostic information for SCA1 progression.

• Changes in ATXN1L complex formation could serve as early indicators of cerebellar dysfunction before clinical symptoms appear.

Therapeutic Target Validation:

• Using ATXN1L antibodies to screen compounds that enhance ATXN1L-Cic interactions, which could theoretically compensate for ATXN1 dysfunction in SCA1.

• Antibody-based high-throughput screening systems to identify molecules that modulate ATXN1L expression or function.

Cellular Pathology Mapping:

• Single-cell applications of ATXN1L antibodies combined with other markers to map cell-specific vulnerabilities in neurodegenerative diseases.

• Spatial transcriptomics approaches incorporating ATXN1L detection to understand regional vulnerability in the cerebellum and other affected brain regions.

Multimodal Imaging:

• Development of imaging agents based on ATXN1L antibody fragments for PET or SPECT imaging of ATXN1L distribution in animal models.

• Correlation of ATXN1L-based imaging findings with functional MRI to link molecular changes to circuit dysfunction.

These emerging applications build on the established role of ATXN1L in compensating for ATXN1 loss-of-function and could significantly advance our understanding of SCA1 pathogenesis while providing new therapeutic avenues.

How can ATXN1L antibodies advance our understanding of its role in immune regulation?

Recent discoveries regarding ATXN1's role in immune regulation suggest that ATXN1L may have similar functions, opening new research avenues where ATXN1L antibodies can provide crucial insights:

B Cell Function Analysis:

• ATXN1L antibodies can help track its expression and localization in B cells, where ATXN1 has been shown to regulate key costimulatory molecules like CD44 and CD80 .

• Chromatin immunoprecipitation using ATXN1L antibodies can identify direct genomic targets in B cells, potentially revealing regulatory networks controlling immune activation.

T Cell Differentiation Studies:

• ATXN1L antibodies can be employed in co-culture experiments to understand how ATXN1L in antigen-presenting cells influences T cell differentiation, particularly Th1 polarization.

• Immunoprecipitation of ATXN1L-containing complexes from immune cells, followed by proteomic analysis, could reveal immune-specific binding partners.

Autoimmunity Models:

• In experimental autoimmune encephalomyelitis (EAE), where ATXN1 deficiency exacerbates disease , ATXN1L antibodies can track potential compensatory changes in ATXN1L expression.

• Flow cytometry applications using ATXN1L antibodies could help identify specific immune cell populations with altered ATXN1L expression in autoimmune conditions.

Signaling Pathway Integration:

• Combining ATXN1L immunodetection with phospho-specific antibodies for ERK and STAT pathways can reveal how ATXN1L integrates with these signaling networks in B cells.

• This approach could help explain the exaggerated proliferation of ATXN1-deficient B cells that has been linked to ERK and STAT pathway activation .

These applications of ATXN1L antibodies in immune research could significantly expand our understanding of ATXN1L beyond its established neurological functions and potentially reveal new therapeutic targets for autoimmune disorders alongside neurodegenerative diseases.

What technological advancements will enhance ATXN1L antibody applications in the future?

Several technological advancements on the horizon will likely transform how ATXN1L antibodies are used in research, enhancing their specificity, application range, and information yield:

Antibody Engineering Innovations:

• Development of recombinant ATXN1L antibodies with enhanced specificity for distinguishing between ATXN1L and ATXN1.

• Creation of bispecific antibodies that simultaneously target ATXN1L and key interaction partners like Cic, allowing for more precise study of functional complexes.

• Single-domain antibodies (nanobodies) against ATXN1L that can penetrate nuclear compartments more efficiently for live-cell imaging applications.

Advanced Imaging Technologies:

• Super-resolution microscopy applications using fluorophore-conjugated ATXN1L antibodies to study nuclear organization of ATXN1L-containing complexes at nanoscale resolution.

• Expansion microscopy protocols optimized for nuclear proteins to visualize ATXN1L distribution relative to chromatin domains.

• Live-cell ATXN1L tracking using cell-permeable antibody fragments to monitor dynamic changes in complex formation.

Single-Cell Applications:

• Integration of ATXN1L antibodies into multiplexed antibody-based single-cell proteomics platforms.

• Combined single-cell transcriptomics and proteomics to correlate ATXN1L protein levels with target gene expression in individual cells.

• Spatial transcriptomics approaches incorporating ATXN1L detection to map its activity across tissue microenvironments.

In Situ Structural Biology:

• Proximity labeling methods using ATXN1L antibodies to map the molecular neighborhood of ATXN1L in intact cells.

• In-cell NMR applications to study structural changes in ATXN1L upon complex formation with partners like Cic.

These technological advances will enable researchers to move beyond current limitations in studying ATXN1L function, potentially revealing new aspects of its role in transcriptional regulation, immune function, and disease pathogenesis. Such advances could be particularly valuable for understanding the context-dependent functions of ATXN1L that have been observed in different experimental systems .