TYK2 Antibody, HRP conjugated

What is TYK2 and why is it important in research applications?

TYK2 (Tyrosine kinase 2) is a non-receptor tyrosine-protein kinase encoded by the TYK2 gene in humans. It may also be identified by alternative names including IMD35, JTK1, and non-receptor tyrosine-protein kinase TYK2. Structurally, the protein has a molecular weight of approximately 133.7 kilodaltons . TYK2 plays a critical role in signal transduction pathways associated with various cytokine receptors, including those for type I and type III interferons, IL-12, IL-23, and IL-10 family cytokines. These pathways are instrumental in immune response regulation, making TYK2 a significant research target for understanding autoimmune disorders, inflammatory conditions, and cancer biology. The protein's function in JAK-STAT signaling pathways has positioned it as a valuable biomarker and potential therapeutic target in numerous disease contexts.

TYK2 research applications extend across multiple disciplines, including immunology, oncology, and inflammatory disease research. Studies typically focus on its phosphorylation states, protein interactions, and expression patterns in different cell types and disease models. The conservation of TYK2 across species, with orthologs in canine, porcine, monkey, mouse, and rat models, further enhances its utility in translational research .

What are the advantages of using HRP-conjugated TYK2 antibodies over unconjugated versions?

HRP-conjugated TYK2 antibodies offer several methodological advantages over unconjugated versions. The direct conjugation of Horseradish Peroxidase (HRP) to the antibody eliminates the need for secondary antibody incubation steps, significantly reducing protocol time, complexity, and potential sources of background signal or cross-reactivity. This streamlined approach is particularly valuable in time-sensitive experiments or when working with limited sample quantities.

The enzymatic activity of HRP provides excellent signal amplification capabilities, enabling detection of even low-abundance TYK2 protein in complex biological samples. When combined with appropriate substrates, HRP generates either colorimetric, chemiluminescent, or fluorescent signals depending on the detection system employed. This versatility allows researchers to select optimal detection methods based on instrumentation availability and sensitivity requirements.

How should TYK2 antibody, HRP conjugated be stored to maintain optimal activity?

Proper storage of HRP-conjugated TYK2 antibodies is essential for maintaining enzymatic activity and antibody binding capacity. The recommended storage temperature for most HRP-conjugated antibodies is at -20°C for long-term preservation, with aliquoting strongly advised to prevent repeated freeze-thaw cycles that can significantly reduce antibody performance. Each freeze-thaw cycle can potentially decrease activity by 10-15%, with substantial degradation occurring after 3-5 cycles.

Monitoring solution clarity before use is essential, as cloudy appearance may indicate protein aggregation and reduced functionality. Most manufacturers recommend using glycerol-based stabilizers (25-50%) for cryopreservation of HRP conjugates, which helps prevent ice crystal formation during freezing. Proper pH maintenance between 6.8-7.4 is also critical for preserving both antibody binding capacity and enzymatic activity of the HRP moiety over time .

What validation assays should be performed before using a new lot of TYK2 antibody, HRP conjugated?

Before implementing a new lot of HRP-conjugated TYK2 antibody in critical experiments, comprehensive validation is essential to ensure performance consistency. Initial titration experiments should establish optimal working concentrations across multiple dilutions (typically 1:500 to 1:5000) using positive control samples with known TYK2 expression levels. This titration should be performed for each specific application (WB, ELISA, IHC) as optimal concentrations may vary between techniques.

Specificity validation is paramount, involving parallel testing with both positive and negative control samples. For Western blotting, this includes lysates from cell lines with confirmed TYK2 expression versus knockout or low-expression lines. The detection of a single band at the expected molecular weight (~133.7 kDa for full-length TYK2) confirms specificity . Cross-reactivity assessment should include testing against related JAK family proteins (JAK1, JAK2, JAK3) to ensure selective TYK2 recognition.

Sensitivity determination involves creating standard curves with recombinant TYK2 protein to establish detection limits. For consistent lot-to-lot comparison, side-by-side testing of the new lot against a previously validated lot using identical samples and protocols is recommended. Researchers should also verify HRP enzymatic activity using standard substrates appropriate for their detection system (TMB, ECL, etc.) and conduct stability tests under experimental conditions to confirm signal consistency over the expected assay duration.

Documentation of all validation parameters, including lot number, dilution factor, incubation conditions, and comparative performance metrics, creates valuable reference data for troubleshooting and experimental reproducibility.

What are the optimal blocking conditions when using HRP-conjugated TYK2 antibodies for Western blotting?

For phospho-TYK2 detection, 3-5% BSA in TBST is strongly recommended as an alternative blocking agent. The duration and temperature of blocking significantly impact results, with optimal conditions typically being 1 hour at room temperature or overnight at 4°C for standard TYK2 detection. Extended blocking periods beyond these recommendations can occasionally mask epitopes and reduce signal intensity.

The membrane washing protocol following blocking is equally important, with 3-5 washes in TBST (5 minutes each) typically sufficient to remove excess blocking agent while preserving specific antibody binding sites. Some researchers report enhanced results using specialized commercial blocking buffers containing synthetic blocking agents particularly when detecting low-abundance TYK2 in complex samples. Regardless of the blocking system selected, maintaining consistency across experiments is essential for reliable data comparison and reproducibility .

How can signal-to-noise ratio be optimized when using TYK2 antibody, HRP conjugated in immunohistochemistry?

Optimizing signal-to-noise ratio for HRP-conjugated TYK2 antibodies in immunohistochemistry requires systematic attention to multiple protocol variables. Antigen retrieval methodology significantly impacts epitope accessibility, with heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) generally providing excellent results for TYK2 detection in formalin-fixed, paraffin-embedded tissues. For tissues with high endogenous peroxidase activity, pre-treatment with 0.3-3% hydrogen peroxide for 10-15 minutes effectively suppresses background signal that would otherwise compromise specific TYK2 detection.

Antibody dilution optimization is essential, with recommended starting ranges of 1:100 to 1:500 for most HRP-conjugated TYK2 antibodies. Systematically testing multiple dilutions on serial sections from the same sample block helps identify the optimal concentration that maximizes specific staining while minimizing background. Incubation conditions significantly impact results, with overnight incubation at 4°C often yielding superior signal-to-noise ratios compared to shorter incubations at room temperature.

The addition of 0.1-0.3% Triton X-100 to antibody diluent can enhance tissue penetration, particularly for nuclear or cytoplasmic TYK2 detection. Signal amplification systems such as tyramide signal amplification (TSA) may be employed for detecting low-abundance TYK2, though careful titration is necessary to prevent signal spillover. Post-staining washes should be thorough (at least 3 × 5 minutes) with agitation to remove unbound antibody effectively.

Including appropriate controls is essential: positive control tissues with known TYK2 expression, negative controls omitting primary antibody, and ideally, TYK2-knockout tissue sections or isotype controls to confirm staining specificity .

What sample preparation techniques ensure optimal TYK2 detection in cell lysates?

Effective sample preparation is fundamental for reliable TYK2 detection in cell lysates, with protocol selection largely dependent on subcellular localization and phosphorylation state of interest. For total TYK2 protein analysis, RIPA buffer (150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris pH 8.0) supplemented with protease inhibitor cocktail provides excellent extraction efficiency while maintaining protein integrity. When investigating phosphorylated TYK2 species, this buffer should be augmented with phosphatase inhibitors (10mM sodium fluoride, 2mM sodium orthovanadate, 1mM sodium pyrophosphate) to preserve phosphoepitopes.

Cell lysis conditions significantly impact TYK2 recovery, with optimal results typically achieved through 30-minute incubation on ice with intermittent vortexing every 5-10 minutes. Sonication (3 × 10 seconds at medium power) following initial lysis enhances extraction of nuclear-associated TYK2 fractions. Post-lysis centrifugation should be performed at 14,000 × g for 15 minutes at 4°C to remove cellular debris while retaining TYK2 in the supernatant.

Freshly prepared lysates demonstrate optimal TYK2 immunoreactivity, though proper storage at -80°C with glycerol addition (10% final concentration) can preserve samples for 3-6 months without significant degradation. Avoiding more than two freeze-thaw cycles is strongly recommended to maintain both total protein and phosphoepitope integrity .

How can TYK2 antibody, HRP conjugated be effectively used in multiplex immunoassays?

Implementing HRP-conjugated TYK2 antibodies in multiplex immunoassays requires careful consideration of detection strategies and potential cross-reactivity issues. For chromogenic multiplexing, HRP-conjugated TYK2 antibodies can be effectively paired with alkaline phosphatase (AP) or glucose oxidase (GO) conjugated antibodies targeting other proteins of interest, with each enzyme utilizing distinct substrates to generate differentiable colors. This approach works particularly well when examining proteins with distinct subcellular localizations.

For fluorescent multiplexing, tyramide signal amplification (TSA) systems compatible with HRP can convert the enzymatic activity to stable fluorescent signals in specific channels. Sequential detection protocols are strongly recommended, with complete inactivation of HRP activity between rounds using 100mM sodium azide or 0.3% hydrogen peroxide for 15-30 minutes. This prevents signal bleed-through that would compromise multiplexing accuracy.

Antibody stripping protocols between sequential detections must be rigorously validated to ensure complete removal of previous detection antibodies without damaging the tissue or cell architecture. Glycine-SDS buffer (pH 2.0) for 10-15 minutes followed by thorough washing often provides effective stripping while preserving sample integrity.

Spectral imaging and linear unmixing approaches have proven particularly valuable when combining HRP-based detection systems with other modalities, allowing computational separation of overlapping signals. For bead-based multiplexed immunoassays, HRP-conjugated TYK2 antibodies can be effectively implemented using distinct bead populations for capturing different analytes, followed by detection with specifically matched HRP-conjugates.

Validation of multiplexed assays should include single-staining controls for each target to confirm that signal intensity and localization patterns remain consistent between single and multiplexed protocols, ensuring that antibody performance is not compromised in the more complex multiplexed environment .

How can phospho-specific TYK2 detection be optimized using HRP-conjugated antibodies?

Detection of phosphorylated TYK2 species presents unique challenges that require specialized methodology when using HRP-conjugated antibodies. Phosphorylation at Tyr1054/1055 within the activation loop represents a critical regulatory mechanism governing TYK2 kinase activity. To preserve these labile phosphoepitopes, sample preparation must include robust phosphatase inhibition combining both serine/threonine (50mM sodium fluoride, 10mM β-glycerophosphate) and tyrosine phosphatase inhibitors (1mM sodium orthovanadate, 1mM sodium molybdate) from the moment of cell lysis.

Stimulation protocols significantly impact phospho-TYK2 detection, with optimal visualization typically requiring cytokine treatment (IFN-α/β, IL-12, or IL-23) for 15-30 minutes prior to lysis. The timing is critical, as phosphorylation peaks between 15-45 minutes post-stimulation before declining due to negative feedback mechanisms. For immunoblotting applications, transferring proteins to PVDF membranes rather than nitrocellulose has been demonstrated to better preserve phosphoepitopes during the transfer process.

BSA (3-5%) is strongly preferred over milk-based blockers for all phospho-TYK2 detection protocols due to the presence of phosphatases in milk that can dephosphorylate target proteins even during brief incubations. When performing Western blot analysis, membrane activation with 100% methanol prior to transfer and the inclusion of 1mM sodium orthovanadate in transfer buffers helps maintain phosphorylation status.

For detecting low-abundance phospho-TYK2 species, immunoprecipitation using total TYK2 antibodies followed by phospho-specific detection can significantly enhance sensitivity. When troubleshooting inconsistent phospho-TYK2 signals, researchers should verify the efficacy of their phosphatase inhibitors using positive control phosphoproteins and consider the impact of cell density, serum starvation conditions, and passage number on basal phosphorylation levels .

What are the key considerations for quantitative analysis of TYK2 expression using HRP-conjugated antibodies?

Quantitative analysis of TYK2 expression using HRP-conjugated antibodies requires meticulous attention to methodology to ensure linear signal response and accurate quantification. Standard curve generation using recombinant TYK2 protein at concentrations spanning 0.1-100 ng/ml establishes the linear detection range for specific experimental conditions. This calibration should be performed under identical conditions to experimental samples, using the same reagent lots, incubation times, and detection parameters.

Signal acquisition timing is critical for accurate quantification, particularly with chemiluminescent substrates where signal intensity changes over time. Capturing images at consistent intervals post-substrate addition (typically 1-5 minutes) minimizes temporal variation. For colorimetric substrates like TMB, stopping the reaction at precisely timed intervals (usually 15-30 minutes) using 2M sulfuric acid ensures measurement consistency.

Digital image acquisition parameters must be optimized to prevent saturation while maintaining sensitivity. For Western blots, multiple exposure times should be captured to ensure measurements fall within the linear dynamic range of detection. Sophisticated analysis software utilizing local background subtraction algorithms typically provides more accurate quantification than simple region-of-interest approaches.

For ELISA-based quantification, four-parameter logistic regression models generally yield superior fitting of standard curves compared to linear regression, particularly at the upper and lower concentration ranges. Technical replicates (minimum triplicate) and biological replicates (minimum n=3) are essential for statistical validity of quantitative TYK2 measurements .

How can cross-reactivity with other JAK family proteins be minimized when using TYK2 antibodies?

Minimizing cross-reactivity with structurally related JAK family proteins (JAK1, JAK2, JAK3) represents a significant challenge when working with TYK2 antibodies due to the high sequence homology within conserved domains. Epitope selection is the primary determinant of specificity, with antibodies targeting unique regions within the FERM or pseudokinase domains typically demonstrating superior TYK2 selectivity compared to those targeting the highly conserved kinase domain.

Pre-absorption techniques can significantly enhance specificity, involving pre-incubation of the HRP-conjugated TYK2 antibody with recombinant JAK1, JAK2, and JAK3 proteins (10-50 μg/ml) for 2 hours at room temperature before application to experimental samples. This process effectively removes antibodies with cross-reactive potential while preserving TYK2-specific binding.

For critical applications requiring absolute specificity confirmation, parallel testing in systems with genetic deletion or knockdown of TYK2 provides definitive validation. CRISPR/Cas9-generated TYK2 knockout cell lines or siRNA-mediated knockdown samples serve as gold-standard negative controls to confirm signal specificity.

When analyzing tissues or cells with co-expression of multiple JAK family members, computational approaches for signal deconvolution can be valuable. This involves quantifying signals from antibodies with known cross-reactivity profiles against recombinant JAK1, JAK2, JAK3, and TYK2, then using matrix mathematics to calculate the true TYK2-specific signal component. While technically demanding, this approach enables accurate TYK2 quantification even with antibodies having modest cross-reactivity issues .

What are effective troubleshooting strategies for weak or absent signals when using TYK2 antibody, HRP conjugated?

When encountering weak or absent signals with HRP-conjugated TYK2 antibodies, a systematic troubleshooting approach addressing each experimental component maximizes the likelihood of resolution. Initial validation of HRP enzymatic activity using direct substrate testing (applying a small volume of properly diluted antibody directly to substrate) confirms conjugate functionality independent of antigen binding. Simultaneously, verification of TYK2 expression in test samples through alternative detection methods (qPCR, mass spectrometry) establishes whether the issue relates to antibody performance or actual protein absence.

Epitope accessibility problems frequently cause false-negative results, particularly with formalin-fixed samples where crosslinking may mask binding sites. Enhanced antigen retrieval protocols, including extended HIER (30-40 minutes) with high-pH buffers (Tris-EDTA, pH 9.0) or enzymatic retrieval using proteinase K (5-20 μg/ml for 10-20 minutes), often recover masked epitopes. For cell lysates, increasing detergent concentration in lysis buffers (up to 1% Triton X-100) improves extraction of membrane-associated TYK2 pools.

Signal amplification systems can overcome sensitivity limitations, with tyramide signal amplification (TSA) providing 10-50 fold enhancement of HRP-generated signals. For Western blotting applications, extended membrane incubation with highly sensitive chemiluminescent substrates (SuperSignal West Femto or Clarity Max) and longer exposure times using cooled CCD cameras significantly improve detection of low-abundance TYK2.

When all optimization attempts fail, epitope competition with endogenous binding partners or post-translational modifications may be responsible. Under these circumstances, sample pre-treatment with mild denaturants (2M urea) or phosphatase treatment (if phosphorylation affects epitope recognition) can expose previously masked binding sites .

How can TYK2 antibody, HRP conjugated be used to study JAK-STAT pathway activation?

HRP-conjugated TYK2 antibodies serve as powerful tools for investigating JAK-STAT pathway activation dynamics in both physiological and pathological contexts. For comprehensive pathway analysis, simultaneous assessment of TYK2 phosphorylation status and downstream STAT protein activation provides mechanistic insights into signal transduction efficiency. Time-course experiments capturing pathway activation at 5, 15, 30, 60, and 120 minutes post-cytokine stimulation reveal the temporal relationship between TYK2 phosphorylation and subsequent STAT activation, with TYK2 phosphorylation typically preceding STAT phosphorylation by 5-15 minutes depending on the specific cytokine and cell type.

Cell type-specific differences in TYK2-dependent signaling can be effectively evaluated through flow cytometry using permeabilized cells labeled with HRP-conjugated TYK2 antibodies followed by fluorescent tyramide signal amplification. This approach allows correlation between surface phenotypic markers and intracellular TYK2 activation status at the single-cell level, revealing potential cellular heterogeneity in response to cytokine stimulation.

For more complex tissue environments, proximity ligation assays combining HRP-conjugated TYK2 antibodies with antibodies against potential interacting partners (cytokine receptors, other JAK family members, or downstream STATs) generate fluorescent signals only when proteins are within 40nm proximity, confirming physical interactions in situ. Quantitative analysis of these interaction events provides spatial and temporal resolution of TYK2-dependent signaling complexes that traditional co-immunoprecipitation approaches cannot achieve .

What are the best practices for studying TYK2 in patient-derived samples using HRP-conjugated antibodies?

Working with patient-derived samples introduces unique challenges for TYK2 analysis that necessitate specialized methodological considerations when using HRP-conjugated antibodies. Sample preservation protocols significantly impact TYK2 detection, with flash-freezing in liquid nitrogen followed by storage at -80°C providing optimal protein integrity for most clinical specimens. For circulating immune cells, immediate isolation and processing are strongly recommended, as delays exceeding 4 hours post-collection result in altered baseline phosphorylation states that can confound interpretation.

Normalization strategies are particularly critical for patient-derived materials where cell composition and protein content may vary substantially between samples. Pathologist-guided tissue microdissection prior to analysis helps isolate regions with comparable cellular composition, reducing confounding variables. For fluid samples like synovial fluid or bronchoalveolar lavage, normalization to total protein concentration following Bradford or BCA quantification ensures comparable loading despite variable cellularity.

Control selection requires careful consideration, with matched healthy donor samples processed identically and simultaneously with patient samples providing the most reliable comparison. Age-matching is particularly important as baseline TYK2 expression and activation patterns demonstrate age-dependent variation, especially in lymphocyte populations.

Batch effects represent a significant concern in clinical studies, necessitating randomized processing of patient and control samples rather than sequential analysis. Including standard reference samples on each experimental run enables inter-experimental normalization to control for day-to-day variation in reagent performance or instrument sensitivity.

For formalin-fixed, paraffin-embedded (FFPE) tissue archives, antigen retrieval protocols require careful optimization, with pressure cooker-based heat-induced epitope retrieval (30 minutes at 110°C in citrate buffer, pH 6.0) typically yielding superior TYK2 detection compared to conventional water bath methods. Importantly, interpreting TYK2 immunoreactivity in FFPE tissues requires consideration of fixation time, as prolonged formalin exposure (>48 hours) may irreversibly mask certain epitopes despite aggressive retrieval methods .

How does TYK2 expression and phosphorylation differ across immune cell populations?

TYK2 expression and phosphorylation patterns exhibit distinctive profiles across immune cell lineages, reflecting cell-specific roles in cytokine responsiveness. Flow cytometric analysis using HRP-conjugated TYK2 antibodies combined with tyramide signal amplification reveals that conventional dendritic cells (cDCs) express the highest levels of total TYK2 protein among peripheral blood mononuclear cells, with approximately 2.3-fold greater expression than CD4+ T cells and 1.7-fold higher than CD14+ monocytes. This elevated expression correlates with the central role of TYK2 in IL-12 and type I interferon signaling critical for dendritic cell function.

Basal phosphorylation states also demonstrate cell type-specific patterns, with CD56bright natural killer cells exhibiting constitutive low-level TYK2 phosphorylation at Tyr1054/1055 even in the absence of exogenous stimulation. This constitutive activation contributes to the heightened cytokine responsiveness characteristic of this NK cell subset. In contrast, naive B cells maintain TYK2 in a predominantly unphosphorylated state until receptor engagement triggers rapid phosphorylation.

Cytokine-induced phosphorylation kinetics reveal functional specialization, with plasmacytoid dendritic cells (pDCs) demonstrating the most rapid and robust TYK2 phosphorylation following type I interferon stimulation (detectable within 2 minutes, peaking at 10-15 minutes), while monocytes show more sustained phosphorylation profiles with maximal activation at 30 minutes post-stimulation that persists for 2-4 hours. T cell subsets display more variable responses, with memory CD4+ T cells phosphorylating TYK2 more rapidly and intensely than naive CD4+ T cells following IL-12 stimulation.

Differentiation state significantly impacts TYK2 expression patterns, with monocyte-derived macrophages upregulating total TYK2 protein approximately 2.5-fold during in vitro differentiation. This upregulation correlates with enhanced responsiveness to IL-10 family cytokines. Interestingly, regulatory T cells (Tregs) maintain comparable total TYK2 expression to conventional CD4+ T cells but demonstrate attenuated phosphorylation responses following cytokine stimulation, reflecting intrinsic differences in proximal signaling regulation that contribute to their distinct functional properties .

What are the implications of TYK2 genetic variants for antibody-based detection methods?

Genetic variation in TYK2 presents significant technical and interpretative challenges for antibody-based detection that researchers must consider when designing experiments and analyzing data. The most prevalent functional variant, P1104A (rs34536443), occurs within the kinase domain and affects protein conformation sufficiently to impact epitope accessibility for certain antibodies. HRP-conjugated antibodies targeting epitopes within amino acids 1090-1120 may demonstrate reduced binding efficiency to the 1104A variant by up to 40% compared to wild-type protein, despite equivalent protein expression levels confirmed by mass spectrometry. This phenomenon can lead to substantial underestimation of TYK2 levels in individuals carrying this variant, which ranges from 4-8% of European populations.

For phospho-specific detection, the I684S variant (rs12720356) located in the pseudokinase domain indirectly affects phosphorylation at the activation loop (Tyr1054/1055) by altering intramolecular regulatory interactions. Consequently, this variant demonstrates approximately 30% reduced phosphorylation following cytokine stimulation despite normal protein expression. Studies employing phospho-specific TYK2 antibodies must therefore account for this variant when comparing patient cohorts or cell lines with different genetic backgrounds.

Rare truncating variants present particular challenges as they may eliminate epitopes while retaining partial protein expression. The specific example of A928fs, which generates a premature stop codon leading to truncation after amino acid 935, eliminates detection with C-terminal-directed antibodies while remaining detectable with N-terminal reagents. This discrepancy has led to contradictory reports regarding TYK2 expression in certain patient cohorts.

For accurate quantification in genetically diverse samples, multiple antibodies targeting distinct, non-variant-affected epitopes should be employed when possible. Alternatively, genotyping key TYK2 variants allows stratified analysis accounting for genetic effects on antibody binding. Quantitative PCR assessment of TYK2 mRNA expression provides a valuable orthogonal measurement unaffected by protein-level variation affecting antibody recognition.

For highest accuracy in population studies, synthetic peptide controls representing major variant sequences can be used to generate correction factors normalizing for differential antibody affinity across variants, enabling more precise comparison between individuals with diverse genetic backgrounds .