TYK2 Antibody, Biotin conjugated

What is TYK2 and why is it significant in immunological research?

TYK2 (Tyrosine kinase 2) is a non-receptor tyrosine kinase and a member of the Janus kinase (JAK) family that plays crucial roles in signal transduction for multiple cytokine receptors. TYK2 has attracted significant attention as a potential therapeutic target for autoimmune diseases due to its involvement in various inflammatory pathways . Research has demonstrated that TYK2 signaling promotes the development of autoreactive CD8+ T cells that express T-BET, a transcription factor essential for cytotoxic T lymphocyte (CTL) development . TYK2's importance stems from its role in mediating signaling for several cytokine receptors, including those for type I interferons, IL-12, and IL-23, making it a central player in both innate and adaptive immune responses.

What are the recommended storage conditions for maintaining optimal activity of biotin-conjugated TYK2 antibodies?

For maximum stability and retention of activity, biotin-conjugated TYK2 antibodies should be stored at -20°C, protected from light. Most commercially available preparations remain stable for approximately one year after shipment when properly stored . For biotin-conjugated antibodies specifically, avoid repeated freeze-thaw cycles as these can accelerate the degradation of both the antibody and the biotin conjugate. Aliquoting upon receipt is recommended for antibodies that will be used multiple times. When working with diluted antibody solutions, storage at 4°C is acceptable for short periods (1-2 weeks), but prolonged storage should be at -20°C with the addition of a carrier protein (0.1% BSA) and preservative to prevent microbial growth and maintain stability.

What validation steps should be performed when using biotin-conjugated TYK2 antibodies in new experimental systems?

When implementing biotin-conjugated TYK2 antibodies in new experimental systems, a systematic validation process is essential:

• Positive and negative controls: Include cell lines known to express TYK2 (e.g., Jurkat cells, RAW 264.7 cells) and those with low/no expression or TYK2 knockout cells .

• Titration experiments: Perform antibody dilution series (typically ranging from 1:50 to 1:1000) to determine optimal concentration for signal-to-noise ratio in your specific application .

• Blocking experiments: Pre-incubate with recombinant TYK2 protein to demonstrate specificity.

• Cross-reactivity assessment: If working with non-human samples, verify species cross-reactivity as documented for the specific antibody clone.

• Comparison with other detection methods: Validate findings using alternative methods such as RT-PCR for mRNA expression or multiple antibody clones recognizing different epitopes.

• Endogenous biotin blocking: For tissues with high endogenous biotin (liver, kidney), implement avidin/biotin blocking steps to reduce background.

• Western blot verification: Confirm antibody specificity by verifying a single band at the expected molecular weight of 134 kDa for TYK2 .

What are the optimal dilution ranges for biotin-conjugated TYK2 antibodies in different applications?

Based on available data for TYK2 antibodies, the following dilution ranges are recommended as starting points, though optimization for specific biotin-conjugated versions is necessary:

Note: These ranges are adapted from data for unconjugated TYK2 antibodies and should be further optimized for biotin-conjugated versions in each specific experimental system.

How can researchers mitigate potential interference from endogenous biotin when using biotin-conjugated TYK2 antibodies?

Endogenous biotin can significantly interfere with biotin-conjugated antibody detection systems, particularly in tissues with high biotin content (liver, kidney, brain) or in cells cultured in biotin-rich media. To mitigate this interference:

• Implement an avidin/biotin blocking step: Before applying the biotin-conjugated TYK2 antibody, block endogenous biotin using commercially available avidin/biotin blocking kits.

• Consider alternative fixation protocols: Some fixation methods can reduce endogenous biotin accessibility.

• Evaluate sample biotin levels: In critical experiments, assess endogenous biotin levels in your experimental system using a streptavidin-only control.

• Biotin-free culture media: For in vitro studies, consider using media formulations with reduced biotin or biotin-depleted serum.

• Alternative detection strategies: In samples with particularly high endogenous biotin, consider using non-biotin detection systems or directly labeled primary antibodies.

• Tissue-specific protocols: For liver samples, which often express high TYK2 levels but also contain substantial endogenous biotin, additional washing steps and extended blocking periods are recommended.

How can biotin-conjugated TYK2 antibodies be utilized to investigate TYK2's role in autoimmune disease models?

Biotin-conjugated TYK2 antibodies offer several sophisticated approaches for investigating TYK2's role in autoimmune disease models:

• Multi-parameter flow cytometry: Biotin-conjugated TYK2 antibodies enable integration into multi-color panels to simultaneously assess TYK2 expression alongside activation markers, cytokine production, and lineage markers in distinct immune cell populations. This is particularly valuable for examining the relationship between TYK2 expression and T-BET levels in CD8+ T cells implicated in autoimmune pathology .

• In situ protein-protein interaction studies: When combined with proximity ligation assays, biotin-conjugated TYK2 antibodies can reveal interactions between TYK2 and other signaling molecules within tissue microenvironments of autoimmune lesions.

• Single-cell analysis: Biotin-conjugated antibodies facilitate sorting and subsequent single-cell analysis of TYK2-expressing cells from disease tissues.

• Intracellular signaling dynamics: These antibodies can be used to track changes in TYK2 phosphorylation status and subcellular localization following cytokine stimulation in cells derived from autoimmune disease models.

• Therapeutic intervention assessment: In models testing TYK2 inhibitors like BMS-986165, these antibodies can help assess target engagement and downstream effects on signaling pathways and immune cell function .

Research has shown that TYK2 signaling promotes the development of autoreactive CD8+ T-BET+ cytotoxic T lymphocytes by mediating IL-12 signaling, and inhibition of TYK2 reduces inflammation in β-cells and prevents onset of autoimmune diabetes in mouse models .

What methodological approaches can resolve discrepancies between total TYK2 protein detection and phosphorylated TYK2 levels in signaling studies?

Discrepancies between total TYK2 and phosphorylated TYK2 (p-TYK2) measurements present significant challenges in signaling studies. To address these methodological issues:

• Sequential detection protocol: Implement a protocol where p-TYK2 is detected first, followed by membrane stripping and re-probing for total TYK2. This approach minimizes epitope masking concerns.

• Parallel sample processing: Process identical samples in parallel, designating specific samples for either p-TYK2 or total TYK2 detection to eliminate stripping-related artifacts.

• Normalization strategy: Calculate the p-TYK2/total TYK2 ratio using densitometric analysis of immunoblots, accounting for loading variations and providing a more accurate representation of activation status.

• Temporal dynamics consideration: Assess both p-TYK2 and total TYK2 across multiple time points following stimulation, as phosphorylation is transient while protein degradation may occur at later timepoints.

• Subcellular fractionation: Separately analyze cytoplasmic and nuclear fractions, as phosphorylated TYK2 may relocate to different cellular compartments during signaling events.

• Complementary techniques: Validate findings using both flow cytometry and immunoblotting, as each technique offers distinct advantages for quantification.

• Phosphatase inhibition: Ensure robust phosphatase inhibition during sample preparation to prevent artificial dephosphorylation during processing.

This systematic approach helps clarify whether observed discrepancies reflect biological phenomena (such as rapid dephosphorylation or protein degradation) versus technical limitations of the detection methods.

How can researchers effectively use biotin-conjugated TYK2 antibodies to investigate the cross-talk between TYK2 and the pseudokinase domain (JH2) in inhibitor studies?

Investigating the complex relationship between TYK2 and its pseudokinase domain (JH2) in the context of novel inhibitor development requires sophisticated experimental approaches:

• Domain-specific co-immunoprecipitation: Use biotin-conjugated TYK2 antibodies targeting different epitopes (JH1 catalytic domain vs. JH2 pseudokinase domain) to pull down protein complexes, followed by mass spectrometry to identify differential binding partners in the presence or absence of JH2-specific inhibitors.

• Conformational change analysis: Implement FRET (Förster Resonance Energy Transfer) assays using biotin-conjugated TYK2 antibodies paired with fluorescently labeled streptavidin alongside antibodies against other domains to detect conformational changes induced by JH2-binding compounds.

• Competitive binding assays: Develop assays where biotin-conjugated TYK2 antibodies compete with potential JH2 ligands, providing insights into binding kinetics and site specificity.

• Functional correlation studies: Correlate the binding of JH2-specific inhibitors (measured via displacement of biotin-conjugated antibodies) with downstream functional outcomes such as inhibition of STAT phosphorylation.

• Structural studies: Use biotin-conjugated antibody fragments to stabilize specific TYK2 conformations for crystallography studies in combination with JH2 ligands.

Research has demonstrated that TYK2 JH2 pseudokinase ligands can effectively inhibit the TYK2 JH1 catalytic domain activity through intermolecular JH2-JH1 interaction, maintaining TYK2 in an inactive conformation . This mechanistic approach differs fundamentally from traditional JAK inhibitors that target the ATP binding site in the JH1 domain, potentially offering greater selectivity and improved safety profiles for therapeutic applications .

What are the most common causes of high background when using biotin-conjugated TYK2 antibodies in immunohistochemistry, and how can they be addressed?

High background is a frequent challenge when using biotin-conjugated antibodies in immunohistochemistry. For TYK2 detection specifically, the following issues and solutions should be considered:

• Endogenous biotin interference:

  • Problem: Particularly problematic in biotin-rich tissues like liver, where TYK2 expression is often studied.

  • Solution: Implement avidin/biotin blocking steps prior to primary antibody incubation; consider alternative detection methods for tissues with extremely high biotin content.

• Inadequate blocking:

  • Problem: Insufficient blocking leads to non-specific binding.

  • Solution: Extend blocking time (1-2 hours at room temperature) using 5-10% normal serum from the same species as the secondary reagent; add 0.1-0.3% Triton X-100 for better penetration.

• Suboptimal fixation:

  • Problem: Overfixation can increase background while underfixation reduces antigen preservation.

  • Solution: Optimize fixation protocols; for TYK2, mild fixation (4% PFA for 15-20 minutes) often yields better results than extended fixation periods.

• Inappropriate antigen retrieval:

  • Problem: TYK2 epitopes may be masked during fixation.

  • Solution: Use TE buffer pH 9.0 for antigen retrieval as specifically recommended for TYK2 detection; alternatively, citrate buffer pH 6.0 may be used .

• Excessive antibody concentration:

  • Problem: Too much antibody increases non-specific binding.

  • Solution: Titrate antibody; start with 1:100 dilution and adjust based on signal-to-noise ratio; for TYK2, dilutions between 1:50-1:500 are typically appropriate .

• Insufficient washing:

  • Problem: Residual unbound antibody contributes to background.

  • Solution: Implement extended washing steps (3-5 washes of 5-10 minutes each) with gentle agitation in PBS-T (0.1% Tween-20).

• Tissue autofluorescence:

  • Problem: Natural tissue fluorescence interferes with detection.

  • Solution: For fluorescent detection systems, use Sudan Black B (0.1-0.3%) treatment post-staining to reduce autofluorescence.

How can researchers differentiate between specific TYK2 detection and cross-reactivity with other JAK family members?

Differentiating between TYK2 and other JAK family members (JAK1, JAK2, JAK3) is critical for accurate interpretation of experimental results. The following methodological approaches help ensure specificity:

• Epitope selection verification: Confirm that your biotin-conjugated TYK2 antibody targets regions with minimal homology to other JAK family members. Antibodies targeting the N-terminal FERM domain or specific regions of the pseudokinase domain typically offer better specificity than those targeting highly conserved kinase domains.

• Knockout/knockdown validation: Include TYK2 knockout or knockdown samples alongside controls for other JAK family members to verify specificity. This is particularly important when studying cell types that express multiple JAK family proteins.

• Competitive binding assays: Pre-incubate the antibody with recombinant TYK2 protein before application to demonstrate that signal reduction is specific to TYK2 blocking.

• Comparative molecular weight analysis: While JAK family members have similar molecular weights, precise SDS-PAGE conditions can resolve them—TYK2 appears at approximately 134 kDa, which can be distinguished from JAK1 (~130 kDa), JAK2 (~125 kDa), and JAK3 (~125 kDa) with high-resolution gels .

• Isoform-specific expression patterns: Leverage known differences in tissue expression patterns—for example, JAK3 expression is largely restricted to hematopoietic cells, while TYK2 is more broadly expressed.

• Selective inhibitor approach: Use selective inhibitors of different JAK family members as experimental controls. TYK2 JH2-targeting compounds exhibit remarkable selectivity over other JAK isoforms in biochemical and cellular assays .

• Mass spectrometry validation: For critical experiments, confirm antibody specificity via immunoprecipitation followed by mass spectrometry to verify the captured protein is indeed TYK2.

What strategies can address poor signal strength when detecting low-abundance TYK2 protein in neurological tissues?

Detecting low-abundance TYK2 in neurological tissues presents unique challenges, particularly given recent interest in TYK2 as a potential target for multiple sclerosis treatment . To overcome these challenges:

• Signal amplification systems:

  • Implement tyramide signal amplification (TSA) systems compatible with biotin-conjugated antibodies

  • Use multilayer detection with additional streptavidin-biotin complexes to build signal layers

• Sample preparation optimization:

  • Extend antigen retrieval time for neurological tissues (20-30 minutes)

  • Consider alternative fixation methods that better preserve TYK2 epitopes in brain tissue

  • Implement tissue clearing techniques for improved antibody penetration in thick sections

• Detection sensitivity enhancement:

  • Use high-sensitivity detection systems (e.g., Qdot-streptavidin conjugates)

  • Employ photomultiplier-based detection systems with increased gain settings

  • Consider computational image processing to enhance signal-to-noise ratio

• Cell-type enrichment:

  • Focus on regions with known TYK2 expression (e.g., microglia-rich areas)

  • Implement laser capture microdissection to isolate specific cell populations before analysis

  • Consider single-cell approaches for heterogeneous neural tissues

• Extended incubation protocols:

  • Increase primary antibody incubation time (overnight at 4°C or up to 48-72 hours)

  • Perform incubations under gentle agitation to improve antibody penetration

• Comparative analysis approach:

  • Include positive control tissues known to express higher TYK2 levels

  • Process MS lesion and non-lesion areas in parallel to identify differential expression

  • Compare with tissues from EAE mouse models where TYK2 signaling has been implicated

Recent research has shown that TYK2 functions as a key neuroimmune modulator capable of regulating inflammatory processes within the brain in autoimmune diseases like MS . TYK2 inhibition reduced pro-inflammatory signaling and microglia inflammatory activity in mouse models, suggesting the importance of detecting even low levels of TYK2 in neurological tissues for understanding its role in disease progression .

How should researchers interpret changes in TYK2 expression levels versus its activation status in disease models?

Proper interpretation of TYK2 data requires distinguishing between expression levels and activation status, as these parameters provide different insights into disease mechanisms:

• Expression level changes:

  • Increased TYK2 expression without corresponding activation may indicate priming of cells for enhanced responsiveness to appropriate stimuli

  • Decreased expression may reflect compensatory downregulation in chronically stimulated systems

  • Tissue-specific expression changes should be interpreted in the context of local cytokine milieu

• Activation status assessment:

  • Phosphorylation at specific residues (particularly in the activation loop) directly correlates with catalytic activity

  • TYK2 can be present but maintained in an inactive state through interaction between its JH2 pseudokinase and JH1 kinase domains

  • Some disease-associated TYK2 variants may show altered baseline activation independent of expression levels

• Integrated interpretation framework:

  • High expression/low activation: Potential negative regulation or requirement for specific activation signals

  • Low expression/high activation: Possible enhanced efficiency of signaling or post-translational modifications increasing activity

  • Correlation with downstream STAT phosphorylation provides functional context for TYK2 status

• Disease-specific considerations:

  • In autoimmune diabetes models, TYK2 activity promotes development of autoreactive CD8+ T-BET+ cytotoxic T lymphocytes through IL-12 signaling

  • TYK2 inhibition in multiple sclerosis models reduces neuroinflammation and microglial activation

  • Changes in TYK2 expression/activation should be examined in relation to disease progression markers

• Temporal dynamics:

  • Acute versus chronic disease stages may show different patterns of TYK2 expression relative to activation

  • Sequential sampling provides more informative data than single timepoint analysis

This nuanced interpretation approach helps distinguish between correlation and causation in disease models and provides clearer direction for therapeutic targeting strategies.

What are the critical considerations when using biotin-conjugated TYK2 antibodies to evaluate the efficacy of TYK2 inhibitors in experimental systems?

When using biotin-conjugated TYK2 antibodies to evaluate TYK2 inhibitor efficacy, researchers should consider these methodological issues:

These methodological considerations ensure robust evaluation of TYK2 inhibitor efficacy and mechanism of action in experimental systems.

How can researchers effectively combine biotin-conjugated TYK2 antibodies with other molecular tools to investigate TYK2's role in neuroinflammatory processes?

To comprehensively investigate TYK2's role in neuroinflammation, researchers should implement integrated approaches combining biotin-conjugated TYK2 antibodies with complementary molecular tools:

• Multi-omics integration strategy:

  • Combine antibody-based TYK2 protein detection with transcriptomics to correlate protein levels with gene expression

  • Integrate phosphoproteomics to map signaling networks downstream of TYK2 in specific neural cell populations

  • Correlate TYK2 activity with metabolomic profiles in neuroinflammatory conditions

• Spatial analysis approaches:

  • Implement multiplexed immunofluorescence with biotin-conjugated TYK2 antibodies alongside markers for microglia, astrocytes, and lymphocyte subsets

  • Use spatial transcriptomics in parallel sections to correlate TYK2 protein localization with gene expression patterns

  • Develop tissue clearing protocols compatible with biotin-conjugated antibodies for 3D visualization of TYK2 distribution in brain tissues

• Functional assessment tools:

  • Combine TYK2 detection with live calcium imaging to correlate TYK2 activity with neural circuit function

  • Implement electrophysiological recordings from regions with differential TYK2 expression

  • Correlate TYK2 levels with measures of blood-brain barrier integrity in MS models

• Cell-specific analysis:

  • Use flow cytometry with biotin-conjugated TYK2 antibodies to analyze TYK2 expression in different CNS-resident and infiltrating immune cells

  • Apply single-cell approaches to understand heterogeneity of TYK2 expression within specific cell populations

  • Develop cell-specific TYK2 knockout models to disambiguate its role in different neural cell types

• Translational correlation:

  • Develop protocols to correlate findings from experimental models with human MS tissue samples

  • Integrate imaging mass cytometry with biotin-conjugated TYK2 antibodies for high-dimensional tissue analysis

  • Correlate findings with clinical outcomes in MS patients with different TYK2 genetic variants

Research has established TYK2 as a key neuroimmune modulator capable of regulating inflammatory processes within the brain in autoimmune diseases . These integrated approaches can help elucidate the precise mechanisms by which TYK2 inhibition reduces pro-inflammatory signaling molecules and microglial inflammatory activity in neuroinflammatory conditions .