TNIK Antibody

What is TNIK and what are its primary functions in cellular biology?

TNIK (TRAF2 and NCK-interacting protein kinase) is a critical serine/threonine kinase that plays significant roles in cellular signaling pathways, particularly in response to environmental stress . It is composed of 1,360 amino acids and features a protein kinase domain essential for its catalytic activity . The protein has a molecular weight of approximately 154.9 kilodaltons . TNIK is expressed ubiquitously throughout the body, with highest expression levels in brain, heart, and skeletal muscle tissues . Functionally, TNIK is involved in the phosphorylation of target proteins, serving as a key regulatory mechanism that influences cell division, apoptosis, and cytoskeletal dynamics . It has been shown to activate the c-Jun N-terminal kinase pathway when overexpressed and can phosphorylate gelsolin, which is the principal intracellular and extracellular actin-severing protein . This suggests TNIK functions in regulating cytoskeletal organization .

Which experimental applications are TNIK antibodies most commonly validated for?

TNIK antibodies have been validated for multiple experimental applications, providing researchers with versatile tools for protein detection and analysis. The most common validated applications include:

• Western Blotting (WB): Nearly all commercial TNIK antibodies are validated for western blot applications, making this the most reliable method for TNIK detection .

• Immunoprecipitation (IP): Several high-quality TNIK antibodies perform well in immunoprecipitation experiments, allowing researchers to isolate TNIK protein complexes from cell lysates .

• Immunofluorescence (IF) and Immunocytochemistry (ICC): TNIK antibodies enable visualization of the protein's subcellular localization through fluorescence microscopy techniques .

• Immunohistochemistry with paraffin-embedded sections (IHC-P): This application allows researchers to examine TNIK expression patterns in tissue sections .

• Enzyme-Linked Immunosorbent Assay (ELISA): Some TNIK antibodies have been validated for quantitative detection in ELISA formats .

The selection of the appropriate application should be guided by experimental objectives and the specific validation data available for each antibody.

What are the species cross-reactivity profiles for most TNIK antibodies?

Based on the search results, TNIK antibodies exhibit varying species cross-reactivity profiles, with the most common reactivity patterns including:

• Human-specific TNIK antibodies: Many commercially available antibodies are specifically developed to detect human TNIK protein .

• Multi-species reactive antibodies: Several TNIK antibodies demonstrate cross-reactivity with human, mouse, and rat TNIK proteins .

• Extended species reactivity: Based on gene homology, some antibodies may potentially recognize TNIK orthologs in canine, porcine, and monkey models, though explicit validation for these species is less common .

When selecting a TNIK antibody for experiments involving non-human models, it is essential to verify the specific cross-reactivity profile through manufacturer validation data or literature references. The high degree of conservation in TNIK protein sequences across mammalian species generally facilitates cross-reactivity, but experimental validation remains crucial for less commonly studied species.

How can researchers distinguish between different TNIK isoforms using antibodies?

TNIK exists in eight isoforms generated through alternative splicing, which contributes to its functional diversity across different cellular contexts . When designing experiments to distinguish between these isoforms:

• Epitope selection: Choose antibodies raised against epitopes that are either common to all isoforms (for total TNIK detection) or unique to specific isoforms (for isoform-specific detection). Antibodies targeting the middle region of TNIK may detect different isoforms than those targeting N- or C-terminal regions .

• Western blot resolution: Use high-resolution SDS-PAGE gels (6-8%) that can effectively separate higher molecular weight proteins to distinguish between isoforms with small molecular weight differences.

• Isoform-specific controls: Include positive controls expressing specific TNIK isoforms to validate antibody specificity and recognition patterns.

• Combined antibody approach: Employ multiple antibodies targeting different TNIK regions to create a comprehensive isoform expression profile through comparative analysis.

• Complementary techniques: Supplement antibody-based detection with RT-PCR or RNA-seq to correlate protein detection with isoform-specific transcript expression.

This multi-faceted approach allows researchers to accurately characterize TNIK isoform expression patterns in their experimental systems.

What are the optimal fixation and antigen retrieval methods for TNIK immunohistochemistry in different tissue types?

While the search results don't provide explicit details on optimal fixation and antigen retrieval methods specifically for TNIK, general principles and available information suggest the following approaches:

• Fixation protocols:

  • For most tissues, standard 10% neutral-buffered formalin fixation for 24-48 hours appears compatible with TNIK detection .

  • For brain tissues, where TNIK shows high expression, 4% paraformaldehyde fixation may preserve antigenicity better while maintaining structural integrity.

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is likely suitable for most TNIK antibodies in paraffin-embedded sections.

  • For tissues with high extracellular matrix content (like heart and skeletal muscle where TNIK is highly expressed), protease-based antigen retrieval or high-pH EDTA buffer (pH 9.0) may improve antibody accessibility.

• Tissue-specific considerations:

  • Brain tissues: Gentle fixation and careful antigen retrieval are essential due to TNIK's critical role in neuronal function .

  • Heart and skeletal muscle: Extended antigen retrieval times may be necessary due to dense tissue architecture .

Researchers should conduct optimization experiments with different fixation durations and antigen retrieval methods for their specific tissue of interest, especially when working with less commonly studied tissues.

How can phospho-specific TNIK antibodies be used to study kinase activation in signaling pathways?

Phospho-specific TNIK antibodies enable researchers to study the activation state of TNIK within complex signaling networks:

• Activation monitoring: Phospho-specific antibodies can detect post-translational modifications that correlate with TNIK's activation state, allowing researchers to monitor kinase activity under different experimental conditions.

• Signaling pathway analysis: Since TNIK has been shown to activate the c-Jun N-terminal kinase pathway , phospho-specific antibodies can help map signaling cascade progression by identifying sequential phosphorylation events.

• Experimental approaches:

  • Paired antibody analysis: Use both phospho-specific and total TNIK antibodies in parallel to calculate the ratio of active to inactive TNIK under different conditions.

  • Immunoprecipitation followed by phospho-detection: Enrich for TNIK protein first, then probe for specific phosphorylation sites to increase detection sensitivity.

  • Phosphatase treatment controls: Include samples treated with phosphatases to confirm phospho-antibody specificity.

• Temporal dynamics: Use phospho-specific antibodies in time-course experiments to track TNIK activation kinetics following stimulation with environmental stressors or pathway agonists.

This approach provides valuable insights into how TNIK's kinase activity is regulated within complex signaling networks and its role in cytoskeletal dynamics and stress responses.

What are the optimal conditions for using TNIK antibodies in Western blotting applications?

Based on the search results and general Western blotting principles for high molecular weight proteins like TNIK (154.9 kDa), the following optimized conditions are recommended:

• Sample preparation:

  • Use RIPA or NP-40 lysis buffers containing protease and phosphatase inhibitors to preserve TNIK integrity.

  • Include reducing agents like DTT or β-mercaptoethanol in sample buffer to ensure complete protein denaturation.

• Gel selection and separation:

  • Use lower percentage (6-8%) polyacrylamide gels to improve resolution of high molecular weight proteins.

  • Consider gradient gels (4-15%) for better separation if analyzing both TNIK and lower molecular weight interaction partners.

• Transfer conditions:

  • Employ wet transfer methods at lower voltage (30V) for extended periods (overnight) to ensure efficient transfer of large proteins.

  • Use PVDF membranes with 0.45μm pore size rather than 0.2μm for better binding of large proteins.

• Antibody dilutions:

  • Most TNIK antibodies perform optimally at dilutions between 1:1000 and 1:10,000 for Western blotting .

  • Cell Signaling Technology recommends 1:1000 dilution for their TNIK antibody .

• Detection optimization:

  • Extended primary antibody incubation (overnight at 4°C) may improve signal quality.

  • Use longer exposure times if working with endogenous TNIK in samples with lower expression levels.

Following these optimized conditions should yield reliable and reproducible TNIK detection by Western blotting.

What troubleshooting approaches are recommended when TNIK antibodies show non-specific binding or weak signals?

When encountering issues with TNIK antibody performance, researchers can implement the following troubleshooting strategies:

• For non-specific binding:

  • Increase blocking intensity: Use 5% BSA or milk in TBS-T for 2 hours at room temperature.

  • Modify washing protocol: Increase number of washes (5-6 times) and duration (10 minutes each).

  • Titrate antibody concentration: Test serial dilutions to identify optimal concentration that maximizes specific signal while minimizing background.

  • Use alternative blockers: Try protein-free blocking buffers if conventional blockers are ineffective.

  • Evaluate secondary antibody: Test different secondary antibodies or reduce secondary antibody concentration.

• For weak signal detection:

  • Enrich target protein: Consider immunoprecipitation before Western blotting for low-abundance samples.

  • Optimize sample preparation: Use phosphatase inhibitors to preserve modifications that might affect epitope recognition.

  • Extend exposure time: For chemiluminescent detection, increase exposure time to capture weak signals.

  • Try signal enhancement systems: Use amplification systems compatible with your detection method.

  • Validate with positive controls: Include samples known to express high levels of TNIK (brain, heart, or skeletal muscle lysates) .

• For inconsistent results:

  • Standardize lysate preparation: Ensure consistent protein extraction efficiency across experimental samples.

  • Check antibody storage conditions: Avoid repeated freeze-thaw cycles that may degrade antibody quality.

  • Validate antibody lot consistency: Test new antibody lots against previous lots when restocking.

Implementing these approaches systematically can resolve many common issues encountered with TNIK antibodies.

How should researchers design controls for validating TNIK antibody specificity in their experimental systems?

Proper controls are essential for confirming TNIK antibody specificity and ensuring reliable experimental results:

• Positive controls:

  • Tissue/cell lysates with known high TNIK expression: Brain, heart, and skeletal muscle tissues show high endogenous TNIK expression and can serve as positive controls .

  • Recombinant TNIK protein: Purified recombinant protein provides a defined standard for antibody validation.

  • Overexpression systems: Cells transfected with TNIK expression vectors create high-expression positive controls.

• Negative controls:

  • TNIK knockout tissues/cells: CRISPR/Cas9 or siRNA-mediated TNIK knockout or knockdown samples provide the most stringent negative controls.

  • Pre-absorption controls: Pre-incubate antibody with immunizing peptide to block specific binding sites before application.

  • Secondary-only controls: Omit primary antibody to assess non-specific binding of secondary detection reagents.

• Validation approaches:

  • Multi-antibody verification: Use multiple antibodies targeting different TNIK epitopes to confirm consistent detection patterns.

  • Orthogonal techniques: Correlate antibody-based results with mRNA expression analysis using qPCR or RNAseq.

  • Functional validation: Confirm antibody detection correlates with expected biological functions or pathway activation.

• Application-specific controls:

  • For immunoprecipitation: Include IgG isotype controls to assess non-specific binding to beads or protein A/G.

  • For immunohistochemistry: Include isotype controls and tissue sections without primary antibody incubation.

Implementing these comprehensive control strategies ensures that experimental results accurately reflect TNIK biology rather than antibody artifacts.

How can TNIK antibodies be used to investigate its role in Wnt signaling pathways and colorectal cancer?

Research has established TNIK as an important regulator in Wnt signaling pathways, with significant implications for colorectal cancer pathogenesis:

• Experimental approaches using TNIK antibodies:

  • Co-immunoprecipitation: TNIK antibodies can be used to pull down protein complexes (using 1:50 dilution as recommended by Cell Signaling Technology) to identify interactions with Wnt pathway components like β-catenin.

  • Chromatin immunoprecipitation (ChIP): TNIK antibodies can help investigate associations with transcriptional complexes at Wnt target gene promoters.

  • Immunohistochemistry: TNIK antibodies enable assessment of expression levels and subcellular localization across normal colon and colorectal cancer tissue specimens .

• Key research applications:

  • Analysis of TNIK phosphorylation status in response to Wnt pathway activation.

  • Correlation of TNIK expression levels with cancer stage and progression.

  • Evaluation of TNIK as a potential therapeutic target through antibody-based detection of inhibitor effects.

  • Investigation of TNIK's role in mediating resistance to existing targeted therapies.

• Methodological considerations:

  • Use multiple antibodies targeting different TNIK domains to distinguish between functionally relevant isoforms.

  • Combine with β-catenin antibodies for co-localization studies in tissue sections.

  • Implement phospho-specific antibodies to track activation state in response to Wnt ligands.

This multi-faceted antibody-based approach can provide valuable insights into TNIK's role in Wnt signaling dysregulation during colorectal cancer development and progression.

What are the considerations for using TNIK antibodies in neuronal tissue analysis and neurodevelopmental research?

TNIK has been implicated in neuronal function and neurodevelopmental processes, making TNIK antibodies valuable tools in neuroscience research:

• Tissue-specific optimization:

  • Fixation protocols should be optimized for neural tissues, with 4% paraformaldehyde generally preferred over harsher fixatives.

  • Antigen retrieval methods may need adjustment due to the delicate nature of neural tissue architecture.

  • Background reduction strategies are particularly important given the complex nature of neural tissue.

• Experimental applications:

  • Immunohistochemistry: TNIK antibodies enable mapping of expression patterns across brain regions and during developmental stages.

  • Synaptic fractionation: Combined with subcellular fractionation, TNIK antibodies can determine enrichment in synaptic compartments.

  • Primary neuronal cultures: Immunocytochemistry applications (at 1:100-1:1000 dilutions) allow visualization of TNIK localization relative to synaptic markers.

• Neurodevelopmental research considerations:

  • Temporal expression analysis: Track TNIK expression changes during critical developmental windows.

  • Co-localization studies: Combine TNIK antibodies with markers for neuronal subtypes, synaptic structures, or cytoskeletal components.

  • Activity-dependent modifications: Investigate TNIK phosphorylation status changes in response to neuronal activity.

• Disease-relevant applications:

  • Several studies have implicated TNIK in neurodevelopmental disorders, making comparative analyses between normal and pathological tissues valuable.

  • TNIK is expressed at high levels in brain tissue , making it an important target for neuroscience research.

Researchers should note that TNIK's role in neuronal function appears to involve complex interactions with cytoskeletal dynamics and synaptic components, requiring careful experimental design and interpretation.

How can TNIK antibodies be integrated into high-throughput screening approaches for drug discovery?

Integrating TNIK antibodies into high-throughput screening (HTS) platforms offers powerful approaches for drug discovery:

• Assay development strategies:

  • ELISA-based screening: Develop sandwich ELISA systems using capture and detection TNIK antibodies to screen compounds that disrupt protein-protein interactions.

  • Phospho-specific detection: Implement phospho-TNIK antibodies in cell-based assays to screen for compounds that inhibit TNIK kinase activity.

  • High-content imaging: Use fluorescently labeled TNIK antibodies in automated microscopy platforms to screen for compounds affecting TNIK subcellular localization.

• Screening methodology optimization:

  • Miniaturization: Adapt TNIK antibody-based detection methods to 384- or 1536-well formats to increase throughput.

  • Automation compatibility: Ensure protocols are compatible with liquid handling robotics and automated incubation/washing systems.

  • Positive controls: Include known TNIK inhibitors or activators as reference standards.

• Validation approaches:

  • Orthogonal assays: Confirm primary hits using alternative TNIK antibody-based assays.

  • Dose-response analysis: Use TNIK antibodies to quantify compound effects across concentration ranges.

  • Target engagement: Employ cellular thermal shift assays (CETSA) with TNIK antibodies to confirm direct compound binding.

• Disease-relevant applications:

  • Cancer therapeutics: Screen for compounds that disrupt TNIK's role in Wnt signaling pathways.

  • Neurological disorders: Identify compounds that modulate TNIK's function in neuronal systems.

  • Anti-inflammatory agents: Discover modulators of TNIK's involvement in immune signaling pathways.

These approaches leverage TNIK antibodies as critical reagents throughout the drug discovery pipeline, from primary screening to mechanism of action studies.

How are TNIK antibodies being used to investigate post-translational modifications and their impact on protein function?

TNIK undergoes various post-translational modifications (PTMs) that regulate its activity and interactions, making PTM-specific antibodies valuable research tools:

• Phosphorylation analysis:

  • Phospho-specific TNIK antibodies enable monitoring of activation states in response to various stimuli.

  • These antibodies can help map kinase-substrate relationships by identifying which kinases phosphorylate TNIK at specific residues.

  • Western blot analysis using phospho-specific antibodies can reveal temporal dynamics of TNIK activation in signaling cascades.

• Additional PTM investigation:

  • Beyond phosphorylation, TNIK may undergo ubiquitination, acetylation, or SUMOylation that affect its stability, localization, or function.

  • Antibodies recognizing these modifications would expand our understanding of TNIK regulation.

• Methodological approaches:

  • Mass spectrometry validation: Combine antibody-based enrichment with mass spectrometry to identify novel PTM sites.

  • Mutagenesis studies: Use antibodies to compare PTM patterns between wild-type and mutant TNIK proteins.

  • Inhibitor studies: Employ PTM-specific antibodies to assess how kinase or phosphatase inhibitors affect TNIK modification status.

• Functional correlation:

  • PTM-specific antibodies help correlate modification patterns with functional outcomes like substrate binding, kinase activity, or protein-protein interactions.

This research area continues to evolve as new PTMs are discovered and their functional significance is elucidated through antibody-based detection methods.

What considerations should be made when using TNIK antibodies in combination with live-cell imaging techniques?

Live-cell imaging applications present unique challenges and opportunities for TNIK investigation:

• Antibody format selection:

  • For live-cell applications, antibody fragments (Fab, scFv) often perform better than full IgG due to improved cell penetration.

  • Fluorescently conjugated antibodies eliminate the need for secondary detection reagents, simplifying protocols .

• Cell delivery strategies:

  • Microinjection: Direct delivery of TNIK antibodies into cells for immediate imaging.

  • Cell-penetrating peptide conjugation: Enhance antibody uptake through CPP fusion.

  • Electroporation: Temporary permeabilization for antibody introduction.

  • Expression of intrabodies: Engineer cells to express intracellular antibodies against TNIK.

• Experimental design considerations:

  • Control experiments must confirm antibody specificity in the live-cell context.

  • Potential functional interference must be assessed, as antibody binding may alter TNIK function.

  • Photobleaching and phototoxicity must be minimized through appropriate imaging parameters.

• Alternative approaches:

  • CRISPR-Cas9 knock-in of fluorescent tags to TNIK may provide a more physiological alternative to antibody-based detection.

  • Fluorescent protein fusions complement antibody-based approaches and can be used in parallel to confirm findings.

While challenging, these approaches can provide unique insights into TNIK dynamics that are not accessible through fixed-cell methods.

How can researchers integrate TNIK antibody-based assays with CRISPR-Cas9 gene editing to study protein function?

Combining TNIK antibodies with CRISPR-Cas9 gene editing creates powerful research strategies:

• Validation frameworks:

  • CRISPR knockout validation: Generate TNIK knockout cell lines to confirm antibody specificity and establish true negative controls.

  • Domain deletion analysis: Create partial TNIK deletions to map epitope locations and functional domains.

  • Epitope tagging: Introduce small epitope tags via CRISPR knock-in to enable detection with highly specific tag antibodies.

• Functional investigation approaches:

  • Structure-function analysis: Use CRISPR to introduce point mutations in key TNIK domains and use antibodies to assess effects on protein-protein interactions or localization.

  • Isoform-specific studies: Selectively disrupt specific TNIK isoforms and use antibodies to confirm isoform-specific knockout.

  • Phosphorylation site mutants: Mutate specific phosphorylation sites and use phospho-specific antibodies to study signaling consequences.

• High-throughput applications:

  • CRISPR screens: Use TNIK antibodies to assess how genetic perturbations affect TNIK expression, modification, or pathway activation.

  • Interaction partner discovery: Combine CRISPR knockout of potential binding partners with co-immunoprecipitation using TNIK antibodies.

• Disease modeling:

  • Patient mutation knock-in: Introduce disease-associated TNIK mutations and use antibodies to study effects on protein function.

  • Therapeutic target validation: Use antibodies to assess efficacy of TNIK-targeted therapies in edited cellular models.

This integrated approach leverages the precision of CRISPR-Cas9 with the detection capabilities of TNIK antibodies to provide comprehensive insights into protein function and disease mechanisms.