TAX1BP3 Antibody

What is TAX1BP3 and why is it important in biological research?

TAX1BP3, also known as Tax-interacting protein 1 (TIP-1) or glutaminase-interacting protein (GIP), is a small PDZ domain-containing protein of 124 amino acids in humans and mice. It is unique in that a single PDZ domain is the only functional and structural unit identified in this protein . TAX1BP3 has gained importance in research due to its roles in:

• Cancer biology (high expression in invasive breast cancer cells)

• Radiation response mechanisms

• Wnt/β-catenin signaling pathway regulation

• Osteogenic and adipogenic differentiation of mesenchymal progenitor cells

Understanding TAX1BP3 function has implications for cancer therapy, bone disorders, and metabolic diseases, making it a significant target for antibody-based research applications .

What applications are TAX1BP3 antibodies suitable for in laboratory research?

Based on vendor specifications and literature reports, TAX1BP3 antibodies have been validated for multiple experimental applications:

Researchers should always optimize antibody concentrations for their specific experimental conditions, as suggested dilutions may vary between different sample types and detection methods .

What tissue and cell line reactivity has been confirmed for TAX1BP3 antibodies?

Published data indicate that TAX1BP3 antibodies show reactivity with:

Tissue samples:

• Human colon tissue

• Human kidney tissue

• Mouse kidney tissue

• Rat kidney tissue

• Bone tissue

• White and brown adipose tissue

Cell lines:

• HeLa cells

• NIH/3T3 cells

• Mesenchymal progenitor cells (including BMSCs and ST2 cells)

Expression levels vary by tissue type, with high expression reported in bone and adipose tissues and moderate expression in heart and skeletal muscle .

How should I design a Western blot experiment to detect TAX1BP3 protein?

For optimal Western blot detection of TAX1BP3:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Load 20-40 μg of total protein per lane

• Gel selection and transfer:

  • Use 12-15% SDS-PAGE gels due to TAX1BP3's small size (14-17 kDa)

  • Transfer to PVDF membranes (0.2 μm pore size) for better retention of small proteins

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary TAX1BP3 antibody (1:500-1:2000 dilution) overnight at 4°C

  • Use appropriate HRP-conjugated secondary antibody

• Detection considerations:

  • The observed molecular weight is typically 15-17 kDa

  • Be aware that the calculated molecular weight (14 kDa) may differ slightly from observed weight due to post-translational modifications

• Controls:

  • Positive controls: HeLa cells, NIH/3T3 cells, or kidney tissue lysates

  • Loading control: β-actin or GAPDH antibodies

What are the best practices for immunohistochemical detection of TAX1BP3?

For successful IHC staining of TAX1BP3:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde

  • Paraffin-embed and section at 4-5 μm thickness

• Antigen retrieval (critical step):

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval (microwave or pressure cooker)

• Antibody protocol:

  • Block endogenous peroxidase with 3% H₂O₂

  • Block non-specific binding with 5-10% normal serum

  • Incubate with TAX1BP3 antibody (1:50-1:500) overnight at 4°C

  • Use appropriate detection system (ABC or polymer-based)

  • Counterstain with hematoxylin

• Validation approach:

  • Human colon and kidney tissues serve as positive controls

  • Include a negative control by omitting primary antibody

How can I optimize TAX1BP3 antibody concentration for my specific application?

A systematic titration approach is recommended:

• For Western blot optimization:

  • Prepare a gradient of antibody dilutions (e.g., 1:500, 1:1000, 1:2000, 1:5000)

  • Use consistent protein loading and identical blotting conditions

  • Select the dilution that provides the best signal-to-noise ratio

• For IHC optimization:

  • Test a range of dilutions (1:50, 1:100, 1:200, 1:500)

  • Assess staining intensity, specificity, and background

  • Consider multiple antigen retrieval methods if initial results are suboptimal

• For ELISA and other applications:

  • Perform checkerboard titration with both capture and detection antibodies

  • Analyze sensitivity and specificity metrics

  • Include appropriate positive and negative controls

• Sample-dependent considerations:

  • Different sample types (cell lines vs. tissues) may require different optimal dilutions

  • Fresh vs. frozen vs. fixed samples may show different antibody sensitivities

How can TAX1BP3 antibodies be used to study radiation-induced membrane translocation?

TAX1BP3/TIP-1 undergoes translocation to the plasma membrane following exposure to ionizing radiation, making it a potential biomarker for radiation response and a target for tumor-specific imaging. To study this phenomenon:

• Experimental setup:

  • Treat cancer cell lines with clinically relevant radiation doses (2-10 Gy)

  • Collect samples at early time points post-irradiation (1-6 hours)

• Subcellular fractionation approach:

  • Separate membrane fractions from cytosolic fractions

  • Perform Western blotting with TAX1BP3 antibodies

  • Include membrane markers (Na⁺/K⁺-ATPase) and cytosolic markers (GAPDH) as controls

• Immunofluorescence microscopy:

  • Fix cells at various timepoints post-irradiation

  • Perform non-permeabilized vs. permeabilized staining to distinguish surface from intracellular TAX1BP3

  • Use confocal microscopy to visualize translocation patterns

• Flow cytometry for quantification:

  • Use non-permeabilized cells to detect surface-expressed TAX1BP3

  • Compare radiation-treated vs. control samples

  • Quantify percentage of positive cells and mean fluorescence intensity

This membrane translocation occurs before the onset of radiation-induced apoptosis and cell death, potentially serving as an early marker of radiation response .

What methods can be used to study TAX1BP3's role in regulating Wnt/β-catenin signaling?

TAX1BP3 has been identified as an inhibitor of β-catenin. To investigate this regulatory role:

• Reporter assay approach:

  • Transfect cells with TOP/FOP luciferase reporter constructs

  • Co-transfect with TAX1BP3 expression vectors or siRNAs

  • Measure Wnt signaling activity with/without Wnt activators (Wnt3a, LiCl)

• Protein interaction studies:

  • Perform co-immunoprecipitation with TAX1BP3 antibodies

  • Analyze β-catenin binding using Western blot

  • Map interaction domains through truncation mutants

• Pathway component analysis:

  • Examine effects of TAX1BP3 overexpression/knockdown on:

    • Non-phosphorylated (active) β-catenin levels

    • LRP6 phosphorylation status

    • GSK3β phosphorylation (Ser9)

  • Monitor nuclear translocation of β-catenin by subcellular fractionation

• Target gene expression analysis:

  • Perform qRT-PCR for Wnt target genes (AXIN2, CCND1, etc.)

  • Validate changes in target protein levels by Western blot

These approaches can help elucidate how TAX1BP3 mechanistically inhibits Wnt/β-catenin signaling in various cellular contexts .

How can TAX1BP3 antibodies be used to investigate osteogenic versus adipogenic differentiation?

Research has shown that TAX1BP3 inhibits osteogenic differentiation while stimulating adipogenic differentiation of mesenchymal progenitor cells. To investigate this role:

• Expression profiling during differentiation:

  • Collect cell lysates at different time points during osteogenic or adipogenic differentiation (days 1, 3, 6, 9)

  • Perform Western blotting with TAX1BP3 antibodies

  • Correlate TAX1BP3 expression with differentiation markers

• Gain/loss of function studies:

  • Overexpress or knock down TAX1BP3 in mesenchymal progenitor cells

  • Induce differentiation using standard protocols

  • Assess outcomes using:

    • Osteogenic markers: ALP activity, Alizarin Red staining, Runx2, osterix

    • Adipogenic markers: Oil Red O staining, PPARγ, C/EBPα, FABP4

• Pathway analysis:

  • Examine Wnt/β-catenin components in differentiation models

  • Assess non-phospho-β-catenin levels

  • Monitor BMP/Smad signaling by checking phospho-Smad1/5 levels

• In vivo confirmation:

  • Use tissue samples from conditional TAX1BP3 knock-in mice

  • Perform IHC with differentiation markers

  • Analyze bone phenotypes (micro-CT) and adipose tissue distribution

These methodologies can clarify how TAX1BP3 regulates the balance between osteogenic and adipogenic differentiation through its effects on key signaling pathways .

What storage and handling conditions are optimal for maintaining TAX1BP3 antibody reactivity?

To preserve antibody activity and prevent degradation:

• Storage recommendations:

  • Short-term (up to 1 week): 4°C

  • Long-term: -20°C in small aliquots to avoid freeze-thaw cycles

  • Avoid more than 5 freeze-thaw cycles

• Buffer considerations:

  • Optimal buffer: PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Some formulations contain BSA (0.1-0.5 mg/ml) for increased stability

• Working solution preparation:

  • Dilute immediately before use in appropriate buffer

  • Keep diluted antibody on ice during experiment

  • Do not store diluted antibody for extended periods

• Shipping and temporary storage:

  • Most TAX1BP3 antibodies are shipped with ice packs

  • Upon receipt, immediately transfer to recommended storage conditions

  • Check for signs of degradation (precipitates, cloudiness)

What are common causes of non-specific binding with TAX1BP3 antibodies and how can they be mitigated?

Non-specific binding can complicate interpretation of results. Common issues and solutions include:

• Background in Western blots:

  • Problem: Multiple bands or high background

  • Solutions:

    • Increase blocking time/concentration (5% milk or BSA)

    • Reduce primary antibody concentration

    • Add 0.1-0.5% Tween-20 to washing buffer

    • Include 0.1% SDS in antibody dilution buffer for polyclonal antibodies

• Non-specific staining in IHC:

  • Problem: Diffuse background staining

  • Solutions:

    • Optimize antigen retrieval (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

    • Block with species-matched normal serum

    • Extend washing steps

    • Consider using monoclonal antibodies for increased specificity

• Cross-reactivity issues:

  • Problem: Unexpected reactivity in negative control samples

  • Solutions:

    • Validate specificity using knockdown/knockout controls

    • Perform peptide competition assays

    • Use alternative antibody clones targeting different epitopes

• Species-specific considerations:

  • Ensure antibody reactivity with your species of interest (human, mouse, rat)

  • Consider sequence homology between species when interpreting results

How should I validate the specificity of TAX1BP3 antibodies for my research?

Rigorous validation is essential for reliable results:

• Positive and negative control samples:

  • Positive controls: HeLa cells, NIH/3T3 cells, kidney tissue

  • Negative controls: Cells with confirmed TAX1BP3 knockdown/knockout

• Molecular weight verification:

  • Confirm detection at expected molecular weight (15-17 kDa)

  • Be aware that post-translational modifications may alter apparent size

• Peptide competition/blocking:

  • Pre-incubate antibody with immunizing peptide

  • Specific signal should be reduced or eliminated

• Orthogonal detection methods:

  • Compare results using different antibodies targeting distinct epitopes

  • Correlate protein detection with mRNA expression data

• Genetic manipulation validation:

  • Analyze samples with TAX1BP3 overexpression

  • Test samples with siRNA/shRNA-mediated knockdown

  • When possible, use CRISPR/Cas9 knockout samples as definitive controls

How can TAX1BP3 antibodies be utilized for cancer research and potential therapeutic applications?

TAX1BP3's roles in cancer biology offer several research avenues:

• Tumor imaging applications:

  • TAX1BP3 is reported to relocate to the cell surface following radiation

  • Antibodies can be adapted for in vivo imaging studies

  • Development of radio-immunoconjugates for tumor-specific imaging

• Targeted therapy approaches:

  • Antibody-drug conjugates targeting surface-expressed TAX1BP3

  • Combination with radiation therapy (radiation-inducible targeting)

  • CAR-T cell development using TAX1BP3 binding domains

• Prognostic biomarker development:

  • Analyze TAX1BP3 expression in tumor microarrays

  • Correlate expression with clinical outcomes

  • Assess association with treatment resistance

• Mechanistic cancer biology:

  • Investigate TAX1BP3's role in Wnt signaling in cancer contexts

  • Study effects on cancer stem cell properties

  • Explore involvement in epithelial-mesenchymal transition

These approaches could potentially establish TAX1BP3 as both a biomarker and therapeutic target in cancer research .

What methodological approaches can be used to study TAX1BP3's PDZ domain interactions?

The PDZ domain is critical for TAX1BP3 function. To investigate its interactions:

• Protein-protein interaction screening:

  • Yeast two-hybrid with PDZ domain as bait

  • Pulldown assays using GST-tagged PDZ domain

  • Proteomics approaches (MS/MS) following immunoprecipitation

• Binding affinity determination:

  • Surface plasmon resonance (SPR) with purified proteins

  • Isothermal titration calorimetry (ITC)

  • Microscale thermophoresis (MST) for quantitative measurements

• Structure-function analysis:

  • Site-directed mutagenesis of key PDZ domain residues

  • Competition assays with known PDZ-binding peptides

  • Structural biology approaches (X-ray, NMR)

• Cellular localization of interactions:

  • Proximity ligation assay (PLA) for in situ detection

  • FRET/BRET analysis of protein-protein interactions

  • Time-lapse imaging to track dynamic interactions

These approaches can help identify novel binding partners and elucidate the molecular mechanisms underlying TAX1BP3's diverse functions .

How can researchers integrate TAX1BP3 antibody-based approaches with other omics technologies?

Modern research benefits from multi-omics integration:

• Proteogenomic approaches:

  • Correlate TAX1BP3 protein levels (antibody-based detection) with transcriptomic data

  • Identify post-translational modifications through IP-MS

  • Map genetic alterations affecting TAX1BP3 expression or function

• Spatial biology applications:

  • Multiplex immunofluorescence with TAX1BP3 and pathway components

  • Spatial transcriptomics combined with protein detection

  • Digital spatial profiling in tissue microenvironments

• Single-cell analysis:

  • Combine flow cytometry with single-cell RNA-seq

  • CyTOF (mass cytometry) panels including TAX1BP3

  • Assess heterogeneity of TAX1BP3 expression in complex tissues

• Functional genomics integration:

  • CRISPR screens affecting TAX1BP3 expression/localization

  • Correlation of genetic dependencies with TAX1BP3 status

  • Synthetic lethality approaches based on TAX1BP3 function

This integrated approach can provide comprehensive insights into TAX1BP3 biology across different biological systems and disease contexts .