ATA7 Antibody

What is ATXN7 antibody and what is its target protein?

ATXN7 antibody specifically recognizes and binds to ataxin-7 protein, a 95.5 kDa protein consisting of 892 amino acid residues in its canonical human form. Ataxin-7 is a member of the Ataxin-7 protein family and functions as a component of the STAGA transcription coactivator-HAT complex. The protein is located in both the nucleus and cytoplasm, with up to three different isoforms reported. Ataxin-7 mediates the interaction of the STAGA complex with CRX (cone-rod homeobox) and participates in CRX-dependent gene activation. Additionally, it plays a necessary role in microtubule cytoskeleton stabilization .

What are the common synonyms and orthologs for ATXN7?

Researchers should be aware of multiple synonyms when searching literature about ATXN7, including OPCA3, SCA7, SGF73, Autosomal dominant cerebellar ataxia with retinal degeneration, SAGA associated factor 73 kDa homolog, and ADCAII. ATXN7 gene orthologs have been documented across various species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken. This cross-species conservation indicates the protein's evolutionary importance and provides opportunities for comparative studies in different model organisms .

What post-translational modifications occur in ATXN7?

ATXN7 undergoes several post-translational modifications that can affect its function and detection by antibodies. The two primary documented modifications include sumoylation (the addition of Small Ubiquitin-like Modifier proteins) and protein cleavage. These modifications can alter the protein's molecular weight, subcellular localization, and functional properties. When designing experiments with ATXN7 antibodies, researchers should consider which modified forms they intend to detect and select antibodies that recognize epitopes either affected or unaffected by these modifications, depending on their experimental goals .

What are the validated applications for ATXN7 antibodies?

ATXN7 antibodies have been validated for several experimental applications, with Western Blot (WB) being the most widely used technique for detecting ATXN7 protein expression levels. Immunocytochemistry/Immunofluorescence (ICC/IF) is another validated application that allows visualization of ATXN7 subcellular localization. Additionally, Enzyme-Linked Immunosorbent Assay (ELISA) provides a quantitative method for measuring ATXN7 protein levels in various sample types. When selecting an ATXN7 antibody for a specific application, researchers should verify that the antibody has been validated for their intended use and sample type .

How should researchers design immunofluorescence experiments with ATXN7 antibodies?

For effective immunofluorescence experiments with ATXN7 antibodies, researchers should follow a methodical approach. Begin with fixation using 100% methanol for 5 minutes, followed by blocking with 1% BSA, 10% normal goat serum, and 0.3M glycine in 0.1% PBS-Tween for 1 hour to prevent non-specific binding. Use appropriate dilutions of primary ATXN7 antibody (typically 1:100 to 1:500, but verify manufacturer recommendations) and incubate overnight at 4°C. After washing, apply fluorescently-labeled secondary antibodies and include DAPI counterstaining to visualize nuclei. Given ATXN7's presence in both nucleus and cytoplasm, careful image acquisition with confocal microscopy is recommended to accurately determine the subcellular distribution pattern .

What methodological considerations are important for Western blot detection of ATXN7?

For optimal Western blot detection of ATXN7, consider these methodological factors: (1) Sample preparation: Use RIPA buffer supplemented with protease inhibitors for efficient extraction of nuclear and cytoplasmic proteins; (2) Protein separation: Given ATXN7's size (95.5 kDa), use 8-10% SDS-PAGE gels for optimal resolution; (3) Transfer conditions: Employ wet transfer at 30V overnight for complete transfer of large proteins; (4) Blocking: Use 5% non-fat milk or BSA in TBST for 1 hour at room temperature; (5) Primary antibody incubation: Dilute ATXN7 antibody appropriately (typically 1:1000 to 1:5000) and incubate overnight at 4°C; (6) Detection: Use HRP-conjugated secondary antibodies with enhanced chemiluminescence detection. Verify results with positive controls such as transfected cell lysates expressing ATXN7 .

How can researchers distinguish between different ATXN7 isoforms?

Distinguishing between the three reported ATXN7 isoforms requires careful experimental design. First, select antibodies with epitopes present in all isoforms for detection of total ATXN7, or isoform-specific antibodies when targeting particular variants. For electrophoretic separation, gradient gels (4-12%) can provide superior resolution of closely migrating isoforms. RT-PCR with isoform-specific primers serves as a complementary approach to confirm protein findings at the mRNA level. Mass spectrometry can definitively identify isoforms through peptide sequencing. When planning experiments involving ATXN7 isoforms, consider potential differential expression across tissues and cell types, as well as functional differences that might influence experimental outcomes and interpretation .

What strategies exist for analyzing ATXN7 interactions with the STAGA complex?

Analyzing ATXN7 interactions with the STAGA transcription coactivator-HAT complex requires sophisticated techniques beyond simple detection. Co-immunoprecipitation (Co-IP) using anti-ATXN7 antibodies can capture the entire complex for subsequent identification of binding partners. Proximity ligation assays (PLA) provide visual confirmation of protein-protein interactions within cells with nanometer resolution. Chromatin immunoprecipitation (ChIP) with ATXN7 antibodies can identify genomic regions where the STAGA complex functions. For quantitative interaction analysis, researchers can employ Förster Resonance Energy Transfer (FRET) or Bioluminescence Resonance Energy Transfer (BRET) between labeled ATXN7 and other STAGA components. When investigating functional consequences, gene expression analysis following ATXN7 knockdown or knockout can identify CRX-dependent genes regulated by this interaction .

How should researchers design experiments to study ATXN7's role in microtubule cytoskeleton stabilization?

To effectively investigate ATXN7's role in microtubule cytoskeleton stabilization, researchers should implement a multi-faceted experimental approach. Begin with co-localization studies using dual immunofluorescence with ATXN7 antibodies and microtubule markers like α-tubulin or β-tubulin. Employ microtubule stability assays, such as cold-induced depolymerization or nocodazole treatment, comparing results between ATXN7-depleted cells and controls. Biochemical fractionation can separate stable (acetylated, detyrosinated) from dynamic microtubule populations, followed by Western blotting to quantify changes. Functional studies should include live-cell imaging with fluorescently-tagged ATXN7 and tubulin to observe dynamic interactions. For definitive mechanistic insights, researchers might perform proximity-dependent biotin identification (BioID) or immunoprecipitation mass spectrometry (IP-MS) to identify ATXN7-interacting proteins involved in microtubule regulation .

How can researchers address discrepancies in ATXN7 antibody detection across different experimental conditions?

When confronting inconsistent ATXN7 antibody detection across experimental conditions, researchers should systematically evaluate multiple factors. First, determine if post-translational modifications like sumoylation or proteolytic cleavage are differentially occurring under various conditions, potentially masking or altering epitopes. Second, compare subcellular fractionation profiles as ATXN7's nuclear-cytoplasmic distribution may vary contextually, affecting extraction efficiency. Third, employ multiple antibodies targeting different ATXN7 epitopes to confirm findings. Fourth, verify antibody specificity through knockdown/knockout validation experiments. Fifth, consider cross-reactivity with structurally similar proteins by performing peptide competition assays. Finally, standardize sample preparation protocols, including lysis buffers, protease inhibitors, and denaturation conditions to minimize technical variability. Comprehensive controls and standardization are essential for resolving discrepancies and ensuring reproducible results .

What analytical approaches should be employed when comparing ATXN7 antibody data between different species?

When comparing ATXN7 antibody data across species, researchers must implement rigorous analytical approaches to ensure valid interpretations. Begin by conducting sequence alignment analysis to determine epitope conservation between species, as ATXN7 orthologs exist in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken. Select antibodies recognizing highly conserved regions when cross-species comparisons are planned. Generate a species-specific calibration curve for quantitative assays, as antibody affinity may vary with species-specific amino acid differences. Employ parallel validation methods in each species under study, including Western blots with species-appropriate positive controls and immunostaining with tissue-specific markers. When analyzing results, normalize expression data to species-specific housekeeping proteins and account for differences in protein extraction efficiency. Finally, complement antibody-based detection with orthogonal techniques like mass spectrometry or RNA-seq to confirm cross-species observations .

How should researchers interpret variations in ATXN7 detection between healthy and disease states?

Interpreting variations in ATXN7 detection between healthy and disease states requires careful consideration of multiple variables. First, establish clear baseline measurements in matched control samples, accounting for potential confounders like age, sex, and tissue type. Quantify both total ATXN7 levels and specific post-translationally modified forms, as disease states may alter modification patterns rather than absolute protein levels. Examine subcellular distribution changes, as pathological conditions might affect nuclear-cytoplasmic trafficking of ATXN7. Correlate ATXN7 changes with functional readouts of STAGA complex activity and CRX-dependent gene expression. When studying neurodegenerative conditions, assess not only protein levels but also potential protein aggregation, which might sequester ATXN7 in insoluble fractions requiring specialized extraction procedures. Implement statistical analyses appropriate for the experimental design, including correction for multiple comparisons when screening numerous disease markers simultaneously .

How might antibody engineering enhance ATXN7 research applications?

Antibody engineering presents transformative opportunities for advancing ATXN7 research beyond conventional applications. Researchers can develop bispecific antibodies that simultaneously target ATXN7 and other STAGA complex components to study protein-protein interactions in situ. Fragment antibodies (Fabs) or single-chain variable fragments (scFvs) offer superior tissue penetration for imaging studies and may access epitopes unavailable to full IgGs. Intrabodies—genetically encoded antibodies expressed inside cells—could enable real-time tracking of ATXN7 dynamics in living systems. Additionally, proximity-labeling antibodies conjugated with biotin ligase enzymes can identify transient ATXN7 interaction partners. For therapeutic applications in SCA7 research, engineered antibodies might target mutant ATXN7 forms while sparing wild-type protein. The integration of these approaches with emerging super-resolution microscopy techniques will provide unprecedented insights into ATXN7's molecular behavior in health and disease .

What methodological adaptations are needed when studying antibody responses in ATXN7 therapeutic development?

Developing ATXN7-targeting therapeutics requires specialized methodological adaptations for analyzing induced antibody responses. Researchers must implement tiered immunogenicity testing similar to anti-drug antibody (ADA) monitoring protocols. This begins with screening assays to detect presence of antibodies against the therapeutic, followed by confirmation assays to validate positive results. For positive samples, characterization studies should determine antibody isotypes, epitope mapping, and binding kinetics. Neutralizing antibody assays are crucial to assess whether induced antibodies impair therapeutic activity. Researchers should design methodologies to distinguish between immunogenicity to the therapeutic versus pre-existing autoantibodies to endogenous ATXN7. Long-term monitoring protocols should evaluate persistence versus transience of antibody responses. Finally, correlation analyses between antibody development, pharmacokinetics/pharmacodynamics (PK/PD), efficacy measures, and adverse events are essential for comprehensive safety and efficacy evaluation .

How can integrating immunoprecipitation with mass spectrometry enhance ATXN7 research?

The integration of immunoprecipitation with mass spectrometry (IP-MS) offers powerful advantages for ATXN7 research beyond traditional antibody applications. This approach allows comprehensive identification of ATXN7 protein complexes, revealing both established and novel interaction partners that may escape detection by targeted methods. IP-MS can characterize post-translational modifications with site-specific resolution, identifying precise locations of sumoylation, phosphorylation, and proteolytic cleavage events that regulate ATXN7 function. Using cross-linking MS (XL-MS) techniques, researchers can map the spatial organization of ATXN7 within the STAGA complex at near-atomic resolution. For proteoform analysis, top-down proteomics following ATXN7 immunoprecipitation can distinguish between different isoforms and modified variants simultaneously. When applied to disease models, this methodology can identify pathological alterations in the ATXN7 interactome. The workflow requires careful optimization of antibody concentration, binding conditions, and elution methods to maximize target enrichment while minimizing non-specific interactions .