TNKS2 Antibody

What is TNKS2 and what is its primary function in cellular processes?

TNKS2 (Tankyrase, TRF1-Interacting Ankyrin-Related ADP-Ribose Polymerase 2) is a poly(ADP-ribose) polymerase that functions in various cellular pathways critical to cell growth and survival. It shares significant homology with Tankyrase 1 (TNKS1), though with some distinct functions. TNKS2 catalyzes the addition of poly(ADP-ribose) chains to target proteins (PARylation), which typically marks them for ubiquitination and subsequent proteasomal degradation .

The primary cellular functions of TNKS2 include:

• Regulation of Wnt/β-catenin signaling through degradation of Axin and other pathway components

• Participation in telomere maintenance

• Involvement in mitotic spindle formation

• Regulation of Notch signaling pathway components

Methodologically, when studying TNKS2 function, it's essential to consider potential functional redundancy with TNKS1 and design experiments that can distinguish between their activities, particularly when using inhibitors that may affect both proteins .

How do I select the appropriate TNKS2 antibody for my specific application?

Selection of a TNKS2 antibody should be guided by your experimental application and specific research questions. Consider these methodological factors:

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IHC, IP, IF, FACS) .

• Epitope location: Choose between:

  • N-terminal antibodies: Better for detecting full-length protein

  • C-terminal antibodies: May detect truncated forms

  • Internal region antibodies: Often provide reliable detection across applications

• Cross-reactivity: Determine if TNKS1/TNKS2 specificity is critical for your research. Some antibodies may cross-react with TNKS1 due to sequence homology. If specificity is crucial, select antibodies raised against less conserved regions .

• Host species: Consider compatibility with other antibodies in multiplex experiments and available secondary detection systems .

• Validation data: Review published literature using the specific antibody clone to verify performance in contexts similar to your experimental design .

For knockout/knockdown validation experiments, compare results between multiple antibodies targeting different epitopes to ensure specificity.

What are the optimal conditions for detecting TNKS2 in Western blotting?

Optimizing Western blot detection of TNKS2 requires attention to several technical factors:

Sample preparation:

• Use RIPA or NP-40 buffer supplemented with protease inhibitors, phosphatase inhibitors, and PARP inhibitors to prevent protein degradation

• Include 1-5 mM DTT or β-mercaptoethanol in lysis buffer

• Keep samples on ice and process rapidly to prevent degradation

Gel electrophoresis and transfer:

• Use 6-8% gels for better resolution (TNKS2 is approximately 127 kDa)

• Transfer at lower voltage (30V) overnight at 4°C for efficient transfer of larger proteins

Antibody conditions:

• Primary antibody: Dilute polyclonal antibodies 1:500-1:2000 in 5% BSA/TBST

• Incubate primary antibody overnight at 4°C

• Include positive controls (cell lines with known TNKS2 expression like H647)

• Include negative controls (TNKS2 knockout/knockdown cells if available)

Detection troubleshooting:

• If background is high, increase blocking time or stringency of washes

• If signal is weak, consider longer exposure times or signal enhancement systems

• To verify specificity, pre-adsorb antibody with the immunizing peptide as a control

Western blot of TNKS2 typically shows a band at approximately 127 kDa, though post-translational modifications may result in higher apparent molecular weights.

How do I differentiate between TNKS1 and TNKS2 functions in my experimental system?

Differentiating between TNKS1 and TNKS2 functions requires specialized methodological approaches:

Genetic manipulation approaches:

• Selective knockdown/knockout: Generate single knockout cell lines for each tankyrase and compare phenotypes. This approach revealed that Notch2 stability is specifically regulated by TNKS1 but not TNKS2 in HEK293T cells .

• Rescue experiments: After TNKS1/2 double knockout, selectively reintroduce either TNKS1 or TNKS2 to determine which can rescue specific phenotypes. This approach helped identify that Notch2 localization is specifically affected by TNKS1 .

Protein interaction analysis:

• Co-immunoprecipitation with isoform-specific antibodies: Use antibodies that specifically recognize unique regions of TNKS1 or TNKS2 .

• Domain swap experiments: Create chimeric proteins with domains from TNKS1 and TNKS2 to identify which domains confer target specificity.

Subcellular localization:
Use immunofluorescence with isoform-specific antibodies to determine if TNKS1 and TNKS2 localize to different subcellular compartments, which may explain functional differences .

Research has demonstrated clear functional distinctions between the isoforms. For example, in HEK293T cells, immunofluorescence analysis showed increased Notch2 staining in TNKS1 knockout cells but not in TNKS2 knockout cells , providing evidence of isoform-specific functions that should be considered when designing TNKS-targeted interventions.

What are the emerging roles of TNKS2 in cancer progression and how can antibody-based detection help characterize these functions?

TNKS2 is increasingly recognized as a key player in cancer progression through multiple mechanisms:

Key oncogenic mechanisms:

• Wnt/β-catenin pathway activation: TNKS2 promotes β-catenin nuclear localization and transcriptional activity by targeting Axin for degradation

• Notch signaling regulation: TNKS2 may influence Notch pathway components, affecting cancer stem cell maintenance

• Telomere maintenance: Contributes to cancer cell immortalization

• Mitotic processes: Affects chromosomal stability and cell division

Antibody-based methodologies for characterizing TNKS2 in cancer:

• Immunohistochemistry (IHC) with patient samples:

  • Compare TNKS2 levels between tumor and adjacent normal tissue

  • Correlate TNKS2 expression with clinical outcomes and progression markers

  • Use multiple antibodies targeting different epitopes to confirm specificity

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions between TNKS2 and cancer-relevant binding partners

  • Visualize interactions in situ within tumor microenvironments

• ChIP-seq following TNKS2 manipulation:

  • Combine with TNKS2 antibodies to identify changes in β-catenin binding to target genes

  • Characterize transcriptional networks affected by TNKS2 activity

• Phospho-specific antibodies:

  • Develop antibodies that detect post-translational modifications of TNKS2

  • Monitor activation status in different cancer contexts

Recent research has demonstrated that TNKS2 promotes lung cancer cell malignancy , suggesting that TNKS2-specific antibodies may have value in diagnostic and prognostic applications for certain cancer types.

How can I optimize immunofluorescence protocols for detecting TNKS2 and its interaction partners?

Optimizing immunofluorescence for TNKS2 detection requires specific technical considerations:

Fixation and permeabilization:

• 4% paraformaldehyde (10-15 minutes at room temperature) preserves most epitopes

• For membrane-associated TNKS2, gentle permeabilization with 0.1% Triton X-100 (5-10 minutes)

• For nuclear TNKS2, use 0.5% Triton X-100 for better nuclear access

Antibody selection and validation:

• Use antibodies validated specifically for immunofluorescence applications

• Validate specificity using TNKS2 knockdown/knockout cells as negative controls

• For co-localization studies, select antibodies from different host species to avoid cross-reactivity

Signal enhancement strategies:

• Tyramide signal amplification for weak signals

• Use high-NA objectives (1.3-1.4) and appropriate filter sets

• Deconvolution or super-resolution microscopy for detailed co-localization studies

Co-localization protocol with β-catenin or Notch2:

• Fix cells in 4% PFA (10 minutes, room temperature)

• Permeabilize with 0.2% Triton X-100 (5 minutes)

• Block with 3% BSA in PBS (1 hour)

• Incubate with anti-TNKS2 (1:100-1:500) and anti-β-catenin or anti-Notch2 (1:100-1:500) in blocking buffer (overnight, 4°C)

• Wash 3× with PBS

• Incubate with appropriate secondary antibodies (1:500, 1 hour, room temperature)

• Counterstain nuclei with DAPI

• Mount with anti-fade medium

For studying Notch2-TNKS interactions, compare patterns between wild-type cells, TNKS inhibitor-treated cells, and γ-secretase inhibitor-treated cells, as these show similar patterns of Notch2 accumulation at the plasma membrane .

What controls should I include when using TNKS2 antibodies in my experiments?

Rigorous control selection is critical for experiments using TNKS2 antibodies:

Essential experimental controls:

• Positive controls:

  • Cell lines with known high TNKS2 expression (e.g., H647 lung cancer cells)

  • Recombinant TNKS2 protein (for Western blots)

  • TNKS2 overexpression systems (e.g., A549 cells with TNKS2 overexpression)

• Negative controls:

  • TNKS2 knockdown/knockout cells generated through RNAi or CRISPR-Cas9

  • Secondary antibody only (no primary antibody) to assess non-specific binding

  • Pre-immune serum (for polyclonal antibodies)

  • Peptide competition assay where antibody is pre-incubated with immunizing peptide

• Specificity controls:

  • TNKS1 knockout cells to assess cross-reactivity with TNKS1

  • Comparison of multiple antibodies targeting different epitopes

  • Use of tagged TNKS2 constructs with independent detection methods

• Technical controls:

  • Loading controls (e.g., β-actin, GAPDH) for Western blots

  • Staining controls (e.g., phalloidin for F-actin) for immunofluorescence

  • Isotype controls matching the primary antibody species and isotype

When working with cancer cell lines, consider including both non-malignant and malignant cells from the same tissue type to establish baseline expression patterns and determine cancer-specific alterations .

How can I effectively use TNKS2 antibodies to study Wnt/β-catenin and Notch signaling pathways?

Studying TNKS2's role in Wnt/β-catenin and Notch signaling requires specialized experimental approaches:

Wnt/β-catenin pathway analysis:

• β-catenin nuclear translocation:

  • Immunofluorescence: Co-stain for TNKS2 and β-catenin, quantify nuclear/cytoplasmic β-catenin ratio

  • Fractionation: Separate nuclear and cytoplasmic fractions, perform Western blots for β-catenin

• Axin stability assessment:

  • Co-immunoprecipitation: Pull down with TNKS2 antibody, blot for Axin

  • Pulse-chase experiments: Measure Axin half-life after TNKS2 manipulation

• Wnt target gene expression:

  • qRT-PCR for target genes (e.g., AXIN2, LEF1, CCND1) after TNKS2 knockdown/overexpression

  • Luciferase reporter assays (TOPFlash/FOPFlash) to measure β-catenin-dependent transcription

Notch signaling analysis:

• Notch receptor processing:

  • Western blot with antibodies specific for cleaved Notch (Val1697) to distinguish between uncleaved and cleaved forms

  • Compare effects of TNKS inhibitors vs. γ-secretase inhibitors on Notch processing

• Notch localization:

  • Immunofluorescence to assess membrane accumulation of Notch2 after TNKS2 manipulation

  • Compare patterns between TNKS1 knockout, TNKS2 knockout, and double knockout cells

• Notch target gene expression:

  • qRT-PCR for Notch targets (e.g., Nestin, HES1, HEY1)

  • Compare expression in wild-type, TNKS knockout, and TNKS-rescued cells

Research has shown that TNKS1 (but not TNKS2) affects Notch2 stability and localization, with membrane accumulation of Notch2 observed in TNKS1 knockout cells . This demonstrates the importance of distinguishing between tankyrase isoforms when studying these signaling pathways.

What are the key considerations for using TNKS2 antibodies in combination with tankyrase inhibitors?

Using TNKS2 antibodies in conjunction with tankyrase inhibitors requires careful experimental design:

Inhibitor selection considerations:

• Specificity spectrum:

  • Most inhibitors target both TNKS1 and TNKS2

  • Some newer compounds may have isoform selectivity

  • Control experiments should include TNKS1/2 knockout cells to distinguish inhibitor effects from off-target effects

• Mode of action:

  • PARP domain inhibitors (e.g., XAV939) block catalytic activity

  • Some newer compounds may target protein-protein interactions or other domains

Experimental design protocols:

• Time-course analysis:

  • Monitor TNKS2 protein levels and localization at multiple timepoints after inhibitor treatment

  • Short-term (minutes to hours): Assess direct enzyme inhibition effects

  • Long-term (12-72 hours): Evaluate compensatory responses and pathway feedback

• Concentration optimization:

  • Perform dose-response curves to identify optimal inhibitor concentrations

  • Include cell viability assays to distinguish specific effects from toxicity

  • Use published IC50 values as starting points, but validate in your specific cell system

• Molecular readouts to assess inhibitor efficacy:

  • PARylation status of known TNKS2 substrates

  • Protein levels of tankyrase targets (should increase with effective inhibition)

  • Wnt/β-catenin pathway activity markers (e.g., nuclear β-catenin, target gene expression)

  • Notch pathway components (e.g., membrane localization of Notch2)

Research has shown that treating cells with tankyrase inhibitor Ti8 leads to accumulation of Notch2 at the plasma membrane similar to treatment with γ-secretase inhibitor DAPT . This suggests tankyrase inhibition may affect Notch processing, which can be monitored using antibodies against both total and cleaved Notch2.

How can mass spectrometry complement antibody-based detection of TNKS2 and its interaction partners?

Mass spectrometry (MS) offers powerful complementary approaches to antibody-based TNKS2 analysis:

Integrated MS-antibody workflows:

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Use TNKS2 antibodies to pull down protein complexes, followed by MS identification

  • This approach identified novel tankyrase targets including HectD1, NKD2, Notch2, Dicer, and Chk2

  • Methodology: Perform IP with anti-TNKS2 antibodies, elute complexes, digest with trypsin, analyze by LC-MS/MS

• Proximity-dependent biotinylation (BioID/TurboID) with MS:

  • Express TNKS2-BioID fusion protein to biotinylate proximal proteins

  • Purify biotinylated proteins using streptavidin and identify by MS

  • Validate hits using co-immunoprecipitation with TNKS2 antibodies

• Post-translational modification mapping:

  • Identify TNKS2 PARylation targets using MS

  • Protocol: Enrich PARylated proteins, remove PAR chains with PARG, identify ADP-ribosylation sites by MS

  • Validate sites using in vitro PARylation assays with recombinant TNKS2

• Quantitative proteomics approach:

  • Compare proteomes of wild-type vs. TNKS2 knockout/knockdown cells

  • Use SILAC, TMT, or label-free quantification

  • Identify proteins with altered abundance as potential TNKS2 targets

The combination of whole proteome analysis of tankyrase knockout cells with traditional antibody-based validation has proven highly effective. For example, researchers identified several novel tankyrase targets including Notch family members through MS, then validated these findings using immunoblot analysis and co-immunoprecipitation with tankyrase antibodies .

What methodological challenges exist when studying TNKS2 in different cancer types, and how can researchers address them?

Studying TNKS2 across different cancer types presents several methodological challenges:

Challenge 1: Variable expression and isoform ratio

• Different cancer types show varying TNKS1:TNKS2 ratios

• Solution: Perform systematic profiling of both tankyrases across cancer cell line panels and patient samples

• Use isoform-specific antibodies and qRT-PCR to establish baseline expression patterns

Challenge 2: Tissue-specific interaction partners

• TNKS2 may interact with different proteins depending on tissue context

• Solution: Perform tissue-specific interactome studies using IP-MS

• Compare TNKS2 binding partners between cancer types to identify common vs. tissue-specific interactions

Challenge 3: Technical issues with immunohistochemistry

• Varying epitope accessibility in different fixation conditions

• Solution: Optimize antigen retrieval protocols for each tissue type

• Validate antibody performance on positive control tissues with known TNKS2 expression

Challenge 4: Interpreting functional outcomes

• The same molecular change may have different consequences in different tissues

• Solution: Use multiple functional readouts (proliferation, migration, apoptosis) as demonstrated in lung cancer studies

• Combine genetic manipulation (knockdown/overexpression) with small molecule inhibitors to distinguish isoform-specific effects

Experimental approach for multi-cancer analysis:

• Generate tissue-specific TNKS2 knockdown and overexpression models

• Perform parallel phenotypic assays (proliferation, migration, apoptosis)

• Compare molecular readouts (β-catenin localization, Notch processing)

• Correlate findings with clinical data on TNKS2 expression and patient outcomes

Recent research with lung cancer cells demonstrates the value of creating both knockdown (H647) and overexpression (A549) models to comprehensively assess TNKS2 function in a specific cancer context .

How can TNKS2 antibodies be used in combination with other techniques to develop potential therapeutic strategies?

TNKS2 antibodies serve as valuable tools in developing therapeutic strategies through several integrated approaches:

Target validation methodologies:

• Expression correlation with clinical outcomes:

  • Use immunohistochemistry with TNKS2 antibodies on tissue microarrays

  • Correlate expression with patient survival, metastasis, and treatment response

  • Identify cancer subtypes most likely to benefit from TNKS2-targeted therapy

• Mechanistic pathway analyses:

  • Combine TNKS2 knockdown/overexpression with antibody-based detection of:

    • Wnt pathway components (β-catenin, Axin)

    • Notch pathway elements (Notch2, Nestin)

    • Telomere maintenance factors

  • Validate key nodes as co-targeting opportunities

Therapeutic development applications:

• Drug screening support:

  • Use TNKS2 antibodies to validate target engagement of candidate compounds

  • Measure changes in TNKS2 substrate levels after treatment

  • Assess pathway modulation through immunoblotting and immunofluorescence

• Combination therapy rationale:

  • Since TNKS knockout cells remain viable, anti-cancer strategies likely require combinational approaches

  • Potential combinations supported by research:

    • TNKS inhibitors + telomerase inhibitors (targeting telomere maintenance)

    • TNKS inhibitors + mitotic inhibitors (targeting spindle defects)

    • TNKS inhibitors + γ-secretase inhibitors (targeting Notch processing)

• Biomarker development:

  • Identify TNKS2-dependent signatures that predict therapy response

  • Develop antibody-based companion diagnostics for patient stratification

  • Monitor treatment efficacy using TNKS2 substrate stability as pharmacodynamic markers

Research has demonstrated that tankyrase inhibition affects multiple cancer-relevant pathways simultaneously, including Wnt/β-catenin and Notch signaling . This multi-pathway effect makes TNKS2 an attractive therapeutic target, particularly in combination strategies that address potential compensatory mechanisms.

What are the emerging methodologies for studying TNKS2 function beyond traditional antibody applications?

Beyond traditional antibody applications, several cutting-edge methodologies are advancing TNKS2 research:

Advanced genetic manipulation techniques:

• CRISPR activation/inhibition (CRISPRa/CRISPRi):

  • Allow for modulating TNKS2 expression without complete knockout

  • Enable temporal control of expression when combined with inducible systems

  • Facilitate isoform-specific targeting through strategic guide RNA design

• Degradation technologies:

  • PROTACs (Proteolysis Targeting Chimeras) targeting TNKS2

  • Auxin-inducible degron (AID) tags for rapid, reversible TNKS2 depletion

  • These approaches complement antibody detection of degradation kinetics

Live-cell imaging innovations:

• FRET/BRET sensors for TNKS2 activity:

  • Design biosensors that change conformation upon PARylation

  • Monitor TNKS2 activity in real-time in living cells

  • Validate sensor accuracy using fixed-cell antibody staining

• Optogenetic control of TNKS2:

  • Create light-inducible TNKS2 activity systems

  • Spatiotemporally control TNKS2 function in specific cellular compartments

  • Complement with antibody-based detection of downstream effects

Single-cell technologies:

• Single-cell proteomics:

  • Analyze TNKS2 expression heterogeneity within tumors

  • Correlate with other pathway components at single-cell resolution

  • Validate findings using multiplexed immunofluorescence

• Spatial transcriptomics with protein detection:

  • Combine spatial gene expression profiles with TNKS2 protein localization

  • Map TNKS2 activity zones within complex tissues

  • Integrate with traditional antibody-based histology

These methodologies complement traditional antibody approaches and are particularly valuable for understanding the complex roles of TNKS2 in dynamic cellular processes and heterogeneous cancer contexts.

What insights has recent research provided about the distinctive functions of TNKS2 compared to TNKS1?

Recent research has revealed several important distinctions between TNKS1 and TNKS2 functions:

Substrate specificity differences:

• Notch2 stability and localization are specifically regulated by TNKS1 but not TNKS2 in HEK293T cells

• Immunofluorescence analysis revealed increased Notch2 staining in TNKS1 knockout but not TNKS2 knockout cells

• This provides a clear example of a protein target showing specificity for one tankyrase isoform in living cells

Developmental roles:

• While double knockout of both tankyrases leads to embryonic lethality in mice, the specific contributions of each isoform to this phenotype are still being elucidated

• The embryonic lethality likely reflects disruption of essential developmental signaling pathways like Wnt/β-catenin and Notch

Subcellular localization:

• Research suggests that TNKS1 may have distinct subcellular localization or unique protein binding partners that promote association with specific substrates like Notch2

• These localization differences may explain some of the observed functional specificity

Cancer-specific roles:

• Emerging evidence suggests tissue-specific roles for TNKS2 in cancer progression

• Recent studies specifically implicate TNKS2 in promoting lung cancer cell malignancy

Methodological implications:

• These findings underscore the importance of isoform-specific approaches in tankyrase research

• For therapeutic development, the distinct functions suggest potential benefits of isoform-selective inhibitors

• When interpreting experimental results, researchers should consider whether observed phenotypes reflect TNKS1, TNKS2, or combined effects

Understanding these distinctions is critical for designing more precise experimental approaches and developing more targeted therapeutic strategies that modulate specific tankyrase functions while minimizing off-target effects.

What are the most reliable antibody validation methods for ensuring TNKS2 antibody specificity?

Ensuring TNKS2 antibody specificity requires comprehensive validation using multiple complementary approaches:

Genetic knockout/knockdown validation:

• CRISPR-Cas9 knockout:

  • Generate complete TNKS2 knockout cell lines

  • Compare antibody signal between wild-type and knockout cells

  • Include TNKS1 knockout and double knockout controls to assess cross-reactivity

• siRNA/shRNA knockdown:

  • Use multiple independent siRNA/shRNA constructs targeting different regions

  • Quantify reduction in signal proportional to knockdown efficiency

  • Control for off-target effects using rescue experiments

Recombinant protein validation:

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be blocked by specific peptide but not unrelated peptides

• Overexpression systems:

  • Express tagged TNKS2 and detect with both anti-tag and anti-TNKS2 antibodies

  • Signal should correlate between both detection methods

  • Test multiple concentrations to evaluate antibody linearity

Cross-platform consistency:

• Multi-technique concordance:

  • Compare detection across Western blot, immunofluorescence, and flow cytometry

  • Consistent results across techniques increase confidence in specificity

  • Different techniques may require different antibody concentrations or conditions

• Epitope mapping:

  • Use antibodies targeting different TNKS2 epitopes

  • Consistent results with multiple antibodies increase confidence

  • Discrepancies may indicate isoform-specific detection or post-translational modifications

Independent method confirmation:

• Mass spectrometry validation:

  • Use immunoprecipitation followed by MS to confirm antibody pulls down TNKS2

  • Quantify ratio of TNKS2 to TNKS1 in immunoprecipitates to assess specificity

• Functional validation:

  • Demonstrate antibody detects changes in TNKS2 after known regulatory events

  • Show expected changes in localization or modification state under specific conditions