axin1 Antibody

What are the optimal applications for AXIN1 antibodies?

AXIN1 antibodies can be effectively used in multiple experimental applications, each requiring specific optimization. Western Blot (WB) represents the most widely documented application, with published evidence supporting its use across various cell lines including HeLa, HT-1080, and HEK-293T cells . For immunohistochemistry (IHC), AXIN1 antibodies have been validated in human colon tissue and mouse stomach tissue, with recommended antigen retrieval using TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0 .

Additional validated applications include immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF/ICC) in cell lines such as A431, and flow cytometry for intracellular detection . When planning experiments, researchers should consider that polyclonal antibodies (e.g., 16541-1-AP) may offer broader epitope recognition while monoclonal antibodies (e.g., 68093-1-Ig) provide consistent reproducibility across experiments, particularly when analyzing specific domains of interest.

How should AXIN1 antibody dilutions be optimized for different applications?

Dilution optimization is critical for maximizing signal-to-noise ratio and ensuring reliable data interpretation. Based on extensive validation data, the following dilution ranges are recommended for specific applications:

These dilutions serve as starting points, and researchers should titrate the antibody in their specific experimental systems to achieve optimal results . Factors affecting optimal dilution include protein expression levels in the sample, detection method sensitivity, and sample preparation methods. For low-expressing samples, lower dilutions (higher antibody concentrations) may be necessary, while highly expressing samples may require higher dilutions to prevent background.

What explains the discrepancy between calculated and observed molecular weights of AXIN1?

AXIN1 exhibits a notable discrepancy between its calculated and observed molecular weights that researchers should account for when interpreting Western blot results. The calculated molecular weight based on amino acid sequence is 92-95 kDa (for 826-862 amino acids), yet the observed molecular weight in SDS-PAGE is consistently higher at 100-120 kDa .

This molecular weight shift is likely attributable to several factors: post-translational modifications such as poly(ADP-ribosyl)ation by tankyrase TNKS and TNKS2 , phosphorylation events that occur during Wnt signaling regulation, and potential conformational characteristics due to AXIN1's numerous functional domains. Additionally, the presence of intrinsically disordered regions, particularly evident in domains outside the structured RGS/APC and DIX/DVL domains, contributes to anomalous migration in gel electrophoresis . When validating antibody specificity, researchers should confirm that detected bands appear within the 100-120 kDa range rather than at the theoretical weight.

What are the recommended storage conditions for maintaining AXIN1 antibody activity?

Proper storage is critical for maintaining antibody reactivity and specificity over time. AXIN1 antibodies should be stored at -20°C, where they remain stable for one year after shipment . The antibodies are provided in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability during freeze-thaw cycles.

Notably, aliquoting is unnecessary for -20°C storage due to the glycerol content, which prevents damage from multiple freeze-thaw cycles . Smaller 20μl packaging sizes typically contain 0.1% BSA for additional stability. When working with the antibody, it's advisable to keep it on ice during experiments and return to -20°C promptly after use. Avoid exposing the antibody to multiple room temperature cycles and contamination to preserve its binding capacity and specificity.

How can researchers study the impact of AXIN1 mutations on β-catenin signaling?

Investigating AXIN1 mutations' effects on β-catenin signaling requires a multi-faceted methodological approach. Recent research analyzed 80 tumor-associated variants and identified 18 that significantly affected β-catenin signaling . This study demonstrates a comprehensive approach researchers can adapt:

First, domain-specific functional assays should be employed to assess mutation effects. Focus on the GSK3β, β-catenin, and RGS/APC interaction domains, which are critical for proper β-catenin regulation . Reporter assays using TCF/LEF luciferase constructs can quantitatively measure downstream transcriptional activation changes resulting from AXIN1 mutations.

Second, co-immunoprecipitation experiments are essential for determining whether mutations disrupt binding to key partners. Most functionally significant mutations were found to lose binding to partners corresponding to the mutated domain . When designing these experiments, researchers should use appropriate controls including wild-type AXIN1 and established binding-deficient mutants.

Third, structural analysis can predict mutation consequences. Using experimentally determined structures (PDB entries 1qz7, 1emu, 1o9u) and AlphaFold predictions for full-length AXIN1, researchers can categorize mutations as interface-located, surface-exposed non-interface, or within the hydrophobic core . This classification helps predict functional impacts based on amino acid properties including size, charge, hydrophobicity, and hydrogen bond capacity.

What methodological approaches reveal relationships between AXIN1 structure and function?

Studying structure-function relationships in AXIN1 requires targeted approaches addressing both structured and intrinsically disordered regions. For the structured domains (RGS/APC and DIX/DVL domains and helical structures binding GSK3β or β-catenin), researchers should employ crystallography or NMR spectroscopy combined with point mutation analysis .

When analyzing truncation mutants, researchers should note that truncated AXIN1 length inversely correlates with β-catenin regulatory function, with longer proteins retaining more functionality . This indicates that even partial truncations impact function, requiring careful experimental design when using deletion constructs.

How should experiments be designed to investigate AXIN1's role in cancer development?

When investigating AXIN1's tumor suppressor role, researchers should employ a comprehensive strategy combining mutation analysis, functional assays, and clinical correlation. AXIN1 mutations are frequently observed in various cancer types, particularly liver cancers, making it essential to distinguish driver from passenger mutations .

For mutation analysis, researchers should focus on the critical GSK3β, β-catenin, and RGS/APC interaction domains, as mutations in these regions are more likely to be functionally significant . When analyzing clinical samples, comprehensive sequencing should be performed to identify co-occurring mutations in other β-catenin regulatory genes such as APC and CTNNB1, as most colorectal and liver cancers carrying AXIN1 missense variants also acquire such mutations .

Cell-based functional analyses should include β-catenin localization studies using immunofluorescence to detect nuclear accumulation, TCF/LEF reporter assays to measure pathway activation, and target gene expression analysis. For in vivo studies, researchers can employ CRISPR-Cas9 technology to introduce specific AXIN1 mutations into cellular or animal models and assess tumorigenic potential.

Bioinformatic approaches using tools like ConSurf for evolutionary conservation analysis can help prioritize variants for functional testing . Such analyses reveal that mutations in highly conserved regions of AXIN1 are more likely to disrupt function than those in less conserved regions.

What factors affect AXIN1 antibody selection for different experimental systems?

Selecting the appropriate AXIN1 antibody requires careful consideration of species reactivity, clone type, and target epitope location. For species reactivity, polyclonal antibody 16541-1-AP has been validated primarily with human samples but has cited reactivity with human, rat, and chicken samples . In contrast, monoclonal antibody 68093-1-Ig offers broader tested reactivity across human, mouse, and rat samples .

The choice between polyclonal and monoclonal antibodies depends on the experimental goals. Polyclonal antibodies like 16541-1-AP offer higher sensitivity by recognizing multiple epitopes, making them advantageous for detecting low-abundance proteins . Monoclonal antibodies such as 68093-1-Ig provide greater specificity and consistency between experiments, beneficial for comparative studies requiring technical reproducibility .

Target epitope location becomes critical when studying particular domains or AXIN1 mutations. Researchers should verify that the antibody epitope doesn't overlap with regions of interest that might be altered or masked in experimental conditions. For interactions studies, epitope mapping can help ensure antibody binding doesn't interfere with protein-protein interactions being investigated.

How can researchers validate AXIN1 antibody specificity in their experimental system?

Robust validation of AXIN1 antibody specificity is essential for generating reliable data. A comprehensive validation approach should include multiple methodologies:

First, perform positive and negative control experiments using cell lines with known AXIN1 expression. The antibody has been positively validated in multiple cell lines including HeLa, HEK-293T, A431, and HepG2 cells . Western blotting should confirm a single predominant band at 100-120 kDa, corresponding to the observed molecular weight of AXIN1 .

Second, knockdown/knockout validation is crucial for confirming specificity. The antibodies have published applications in knockdown/knockout experiments that can serve as reference protocols . Signal reduction or elimination following AXIN1 siRNA treatment or CRISPR-Cas9 knockout provides compelling evidence of specificity.

Third, cross-validation using multiple detection methods strengthens confidence in antibody performance. If a protein is detected by Western blot, confirmatory immunofluorescence or immunohistochemistry with the same antibody should demonstrate consistent localization patterns. AXIN1 typically shows cytoplasmic localization with potential membranous or nuclear distribution depending on cellular context and Wnt pathway activation status.

What are the critical steps for successful Western blot detection of AXIN1?

Western blot detection of AXIN1 requires attention to several critical parameters. Protein extraction methods significantly impact results - use RIPA buffer supplemented with protease and phosphatase inhibitors to preserve post-translational modifications that affect AXIN1 migration and antibody recognition. Given AXIN1's high molecular weight (100-120 kDa), use lower percentage gels (8-10% acrylamide) for better resolution and longer transfer times to ensure complete transfer to membranes.

For primary antibody incubation, recommended dilutions range from 1:1000-1:6000 for polyclonal antibody 16541-1-AP and 1:5000-1:50000 for monoclonal antibody 68093-1-Ig . Overnight incubation at 4°C typically yields better results than shorter incubations at room temperature. When troubleshooting weak signals, consider sample enrichment through immunoprecipitation before Western blotting or using enhanced chemiluminescence detection systems.

To improve reproducibility, include positive control lysates from cells with known AXIN1 expression such as HeLa, HT-1080, or HEK-293T cells . For protein loading control selection, choose controls that don't overlap with AXIN1's molecular weight range to avoid masking the target signal.

How should researchers optimize immunohistochemistry protocols for AXIN1 detection?

Successful immunohistochemical detection of AXIN1 depends on optimized tissue preparation and antigen retrieval. For tissue fixation, 10% neutral buffered formalin is recommended, with fixation time optimized to preserve antigenicity while maintaining morphology. Paraffin embedding should follow standard protocols with careful temperature control to avoid protein denaturation.

Antigen retrieval is critical for AXIN1 detection. The recommended protocol employs TE buffer at pH 9.0, with citrate buffer at pH 6.0 as an alternative . Heat-induced epitope retrieval using pressure cooking or microwave methods typically yields better results than enzymatic retrieval for AXIN1. The optimal antibody dilutions differ significantly between polyclonal (1:50-1:500) and monoclonal (1:1000-1:4000) antibodies , necessitating empirical determination for each tissue type.

For visualization systems, 3,3'-diaminobenzidine (DAB) detection works well for AXIN1, but tyramide signal amplification may improve sensitivity for tissues with low expression. Counterstaining should be optimized to provide sufficient contrast without obscuring specific staining. Validation should include both positive control tissues (human colon tissue for polyclonal antibody, mouse stomach tissue for monoclonal antibody) and negative controls using isotype-matched non-specific antibodies.

How can AXIN1 antibodies advance understanding of Wnt signaling in disease contexts?

AXIN1 antibodies offer powerful tools for investigating Wnt signaling dysregulation across multiple disease contexts. In cancer research, these antibodies enable precise characterization of AXIN1 mutations and their impact on β-catenin regulation. Recent studies have identified that only a subset of missense mutations significantly affect β-catenin signaling, while all truncating mutations at least partially impair this function .

For mechanistic studies, AXIN1 antibodies facilitate investigation of protein-protein interactions within the β-catenin destruction complex. Co-immunoprecipitation experiments using AXIN1 antibodies can reveal how mutations disrupt binding to partners like GSK3β, β-catenin, and APC . This approach can be extended to study interactions in non-cancer contexts like developmental disorders and degenerative diseases where Wnt signaling plays crucial roles.

Emerging single-cell technologies combined with AXIN1 immunodetection can reveal cell-type specific responses to Wnt pathway modulation in heterogeneous tissues. This approach could be particularly valuable for understanding tissue-specific effects of AXIN1 dysfunction, potentially explaining why AXIN1 mutations predominantly drive certain cancer types like hepatocellular carcinoma despite AXIN1's ubiquitous expression.

What emerging technologies could enhance AXIN1 antibody applications in research?

Several cutting-edge technologies promise to expand AXIN1 antibody applications in research. Proximity ligation assays using AXIN1 antibodies can provide spatial resolution of protein-protein interactions in situ, revealing where in the cell AXIN1 complexes form under different conditions. This approach could help elucidate how the subcellular localization of AXIN1 complexes changes during Wnt pathway activation or in disease states.

Mass spectrometry immunoprecipitation techniques using AXIN1 antibodies can identify novel interaction partners and post-translational modifications. Given AXIN1's role as a scaffold protein and its poly(ADP-ribosyl)ation by tankyrase TNKS and TNKS2 , comprehensive characterization of its interactome and modifications could reveal new regulatory mechanisms and therapeutic targets.

CRISPR-Cas9 gene editing combined with AXIN1 antibody-based detection methods enables precise correlation between genotype and phenotype. By introducing specific mutations identified in patient samples and monitoring resulting changes in AXIN1 complex formation and function, researchers can establish causal relationships between genetic variants and disease mechanisms, particularly in cancer development where AXIN1 functions as a tumor suppressor .