ATAB2 Antibody

What is ATAD2 and what are its primary biological functions?

ATAD2 (ATPase family AAA structural domain-containing protein 2) is a cancer testicular protein involved in multiple cellular signaling pathways. It plays crucial roles in DNA replication, transcription, and cell cycle control activities that are fundamental to cell survival . The protein contains two "druggable" structural domains - a bromodomain and an ATPase structural domain - making it a potential therapeutic target . ATAD2 is particularly notable for its involvement in cancer biology, where it functions as a transcriptional coactivator and chromatin regulator affecting gene expression patterns associated with cell proliferation.

What is TAB2 and what cellular processes does it regulate?

TAB2 (TGF-beta activated kinase 1 binding protein 2) functions as a binding partner for MAP3K7 (also known as TAK1), mediating various signaling cascades. The canonical human TAB2 protein comprises 693 amino acid residues with a molecular mass of 76.5 kDa . It localizes to the membrane, lysosomes, and cytoplasm, with significant roles in cardiac tissue development and autophagy pathways . TAB2 undergoes various post-translational modifications including methylation, ubiquitination, and phosphorylation that regulate its activity . Alternative names include MAP3K7IP2, CHTD2, TAK1-binding protein 2, and mitogen-activated protein kinase kinase kinase 7-interacting protein 2 .

How is ATAD2 expression linked to cancer pathology?

ATAD2 overexpression has been documented in multiple cancer types, including:

• Lung adenocarcinoma

• Breast cancer

• Colorectal cancer

• Gastric cancer

• Hepatocellular tumors

• Ovarian cancer

• Cervical cancer

• Endometrial cancer

In endometrial cancer specifically, high ATAD2 expression correlates with advanced FIGO stage, poor pathological grading, extensive lymph node infiltration, deep myometrial infiltration, and increased recurrence rates . These correlations strongly suggest that ATAD2 functions as an oncogenic driver promoting tumor aggression and progression.

What criteria should researchers use when selecting anti-ATAD2 or anti-TAB2 antibodies?

When selecting these antibodies, researchers should evaluate:

• Validation methods: Look for antibodies validated through multiple applications (e.g., Western blot, IHC, ICC-IF)

• Epitope location: Consider whether the antibody targets functionally relevant domains

• Clone type: Determine whether monoclonal specificity or polyclonal broad reactivity better suits your application

• Species reactivity: Verify cross-reactivity with your experimental model organism

• Publication record: Prioritize antibodies with documented use in peer-reviewed research

For ATAD2 antibodies, those targeting either the bromodomain or ATPase domain may provide insights into specific functional aspects. For TAB2, consider antibodies that can distinguish between the two reported protein isoforms if your research question addresses isoform-specific functions .

How can researchers definitively validate antibody specificity for ATAD2 or TAB2?

Multi-parameter validation approaches should include:

• Knockout/knockdown controls: Compare staining patterns between wild-type samples and those where the target protein has been depleted

• Peptide competition assays: Pre-incubation with immunizing peptide should abolish specific signals

• Orthogonal detection methods: Correlation of antibody staining with mRNA expression data

• Western blot molecular weight verification: Confirm single band at expected molecular weight (76.5 kDa for TAB2 , appropriate weight for ATAD2)

• Recombinant protein controls: Use purified proteins as positive controls

For TAB2 specifically, researchers can verify expected subcellular localization patterns in membrane, lysosomes, and cytoplasm compartments as described in product documentation .

What are the optimal applications for anti-ATAD2 antibodies in cancer research?

Based on published research, anti-ATAD2 antibodies have proven valuable in:

• Immunohistochemistry (IHC): For detecting expression levels in primary tumors and correlating with clinicopathological parameters

• Western blot analysis: For quantifying protein levels and evaluating knockdown efficiency

• Chromatin immunoprecipitation (ChIP): For investigating ATAD2's role in transcriptional regulation

• Co-immunoprecipitation: For identifying protein interaction partners, particularly with transcription factors like E2F1, E2F2, and MYBL2

When designing ATAD2-focused experiments, researchers should consider incorporating analysis of associated pathways, particularly angiogenesis markers and immune infiltration parameters, as ATAD2 has demonstrated roles in promoting tumor growth, angiogenesis, and influencing immune cell infiltration in endometrial cancer .

What experimental parameters need optimization when using TAB2 antibodies in flow cytometry?

For flow cytometry applications with TAB2 antibodies, researchers should optimize:

• Fixation protocol: PFA fixation (2%) followed by permeabilization with Triton X-100 (0.5%) has been documented for successful detection

• Antibody dilution: Begin with 1:100 dilution for primary antibody incubation (1 hour at room temperature) as a starting point

• Secondary antibody selection: Anti-rabbit IgG conjugated to appropriate fluorophores (e.g., AlexaFluor 488) at 1:1000 dilution

• Proper controls: Include isotype controls (e.g., unimmunized rabbit IgG) to establish background fluorescence levels

• Gating strategy: Design to exclude debris, doublets, and dead cells before analyzing target protein expression

How should researchers design experiments to investigate ATAD2's role in tumor angiogenesis?

A comprehensive experimental approach should include:

• In vitro angiogenesis assays:

  • Endothelial tube formation assays using conditioned media from ATAD2-manipulated cancer cells

  • Endothelial cell migration and proliferation assays

• In vivo models:

  • Xenograft models with ATAD2 overexpression or knockdown

  • Analysis of microvessel density using CD31/CD34 staining

  • Contrast-enhanced imaging to assess tumor vasculature

• Molecular analyses:

  • Evaluation of angiogenic factor expression (VEGF, bFGF, angiopoietins)

  • Analysis of ATAD2 co-expressed genes through bioinformatic approaches like GO and KEGG enrichment analyses

  • Correlation between ATAD2 expression and immune infiltration using tools like TISIDB, TIMER, and GEPIA

• Clinical correlation:

  • Analysis of ATAD2 expression in relation to tumor vascularization in patient samples

What bioinformatic tools are most effective for analyzing ATAD2 co-expression networks?

For comprehensive analysis of ATAD2-associated gene networks, researchers should employ:

• UALCAN: Effective for screening ATAD2 co-expressed genes across cancer datasets

• Gene Ontology (GO) analysis: For functional categorization of co-expressed genes

• Kyoto Encyclopedia of Genes and Genomes (KEGG): For pathway enrichment analysis

• Sangerbox: For performing integrated enrichment analyses of identified gene sets

• Immune System Interaction and Drug Bank (TISIDB): For assessing correlations between ATAD2 and immune infiltration

• TIMER and GEPIA: For additional validation of immune infiltration associations

These tools collectively provide a systems biology approach to understanding ATAD2's broader functional impact beyond its direct molecular interactions.

How can researchers effectively characterize antibody-epitope interactions for TAB2 antibodies?

Based on advanced epitope mapping studies, researchers can employ:

• Combinatorial peptide libraries: Analysis of hexapeptide mixtures (as demonstrated with TGF-alpha antibody tAb2) can identify high-affinity binding motifs

• Comparison with phage display: Chemical peptide libraries have demonstrated superior ability to identify multiple high-affinity binding peptides compared to phage display, which may identify only dominant binding sequences

• Structural variation analysis: Investigating how different amino acid substitutions affect antibody binding to determine key residues for interaction

• Induced fit mechanism assessment: Examine the structural basis of antibody-peptide recognition, particularly when high variability of binding sequences is observed

This comprehensive approach provides detailed insights into the structural basis of antibody-antigen interactions and can guide epitope-specific applications.

What are common causes of non-specific binding with ATAD2 and TAB2 antibodies, and how can they be mitigated?

Common issues and solutions include:

How can researchers optimize immunohistochemical detection of ATAD2 in formalin-fixed paraffin-embedded (FFPE) tumor samples?

For optimal ATAD2 detection in FFPE samples:

• Antigen retrieval optimization:

  • Compare heat-induced epitope retrieval methods (citrate vs. EDTA buffers)

  • Test pH variations (pH 6.0 vs. pH 9.0) to maximize epitope accessibility

• Signal amplification strategies:

  • Polymer-based detection systems for enhanced sensitivity

  • Tyramide signal amplification for low-abundance detection

• Counterstaining considerations:

  • Light hematoxylin counterstaining to avoid obscuring nuclear ATAD2 signals

  • Digital image analysis for quantitative assessment of staining intensity

• Validation controls:

  • Include known positive and negative tissue controls in each staining run

  • Use tissues with gradient expression to calibrate scoring systems

• Correlation with clinical parameters:

  • Establish standardized scoring systems based on staining intensity and percentage of positive cells

  • Correlate with parameters like FIGO staging, lymph node status, and myometrial infiltration

How can ATAD2 antibodies be utilized to evaluate potential therapeutic targets in cancer?

ATAD2 antibodies offer multiple applications in therapeutic development:

• Target validation studies:

  • Immunoprecipitation followed by mass spectrometry to identify interaction partners

  • ChIP-seq to map genome-wide binding sites and identify regulated genes

• Drug screening applications:

  • Competitive binding assays to evaluate small molecule inhibitors targeting the bromodomain or ATPase domain

  • Cell-based assays measuring ATAD2 displacement from chromatin

• Predictive biomarker development:

  • Stratification of patient samples based on ATAD2 expression levels

  • Correlation of expression with response to existing therapies

  • Development of companion diagnostics for emerging ATAD2 inhibitors

• Mechanism of action studies:

  • Analysis of changes in ATAD2 localization, post-translational modifications, or protein interactions following drug treatment

  • Evaluation of downstream transcriptional changes upon ATAD2 inhibition

The "druggable" nature of ATAD2's structural domains makes antibodies valuable tools for characterizing potential therapeutic interventions .

What emerging applications exist for studying TAB2's role in cardiac development using specific antibodies?

TAB2 antibodies enable several advanced approaches to cardiac research:

• Developmental timing studies:

  • Immunohistochemical analysis of TAB2 expression in ventricular trabeculae, endothelial cells of conotruncal cushions, and developing aortic valves during embryogenesis

  • Temporal correlation with cardiac morphogenesis milestones

• Lineage-specific analyses:

  • Co-localization studies with endothelial, myocardial, and valve progenitor markers

  • Flow cytometry-based sorting of TAB2-expressing cardiac progenitor populations

• Signaling pathway integration:

  • Investigation of TAB2's interactions with TGF-β/BMP signaling components in cardiac development

  • Analysis of its role in cardiac autophagy pathways through co-localization with autophagy markers

• Congenital heart disease models:

  • Characterization of TAB2 expression patterns in animal models of cardiac malformations

  • Correlation of TAB2 variants with human congenital heart defects

These approaches provide mechanistic insights into TAB2's developmental functions beyond its better-characterized immune signaling roles.