Phospho-TYK2 (Tyr1054) Antibody

What is Phospho-TYK2 (Tyr1054) Antibody and what does it specifically detect?

Phospho-TYK2 (Tyr1054) Antibody is a rabbit polyclonal antibody specifically designed to detect TYK2 (Tyrosine Kinase 2) protein only when phosphorylated at tyrosine residue 1054. This antibody provides high specificity for monitoring the activation state of TYK2 in experimental systems, as phosphorylation at Tyr1054 represents a critical regulatory event in TYK2 kinase activity . The antibody is typically produced by immunizing rabbits with synthetic phosphopeptides corresponding to amino acid sequences surrounding the phosphorylated Tyr1054 residue of human TYK2. Following immunization, the antibodies are purified using affinity chromatography with epitope-specific phosphopeptides, and non-phospho-specific antibodies are typically removed through additional chromatography steps using non-phosphopeptides . This rigorous production process ensures that the antibody specifically recognizes the phosphorylated form of TYK2 without cross-reactivity to the non-phosphorylated protein, making it an invaluable tool for investigating TYK2 activation in signaling pathway studies.

How does Phospho-TYK2 (Tyr1054) Antibody differ from Phospho-TYK2 (Tyr1054/1055) Antibody?

The principal distinction between these two antibodies lies in their epitope recognition patterns and the biological significance of the phosphorylation events they detect. Phospho-TYK2 (Tyr1054) Antibody recognizes TYK2 protein when phosphorylated solely at tyrosine residue 1054, providing researchers with the ability to monitor this specific modification event . In contrast, Phospho-TYK2 (Tyr1054/1055) Antibody detects TYK2 only when phosphorylated at both adjacent tyrosine residues (1054 and 1055), representing a distinct activation state of the protein . Biologically, the dual phosphorylation at Tyr1054 and Tyr1055 is particularly significant as it induces complete kinase activation, as noted in the literature: "Phosphorylation by JAK1 at Tyr-1054 and Tyr-1055 induces kinase activation" . When selecting between these antibodies, researchers should consider their specific experimental questions—whether investigating intermediate phosphorylation states (single site at Tyr1054) or fully activated TYK2 (dual phosphorylation). These distinct antibodies provide complementary tools for dissecting the sequential phosphorylation events in TYK2 activation and their respective functional outcomes in signaling cascades.

What are the technical specifications of commercially available Phospho-TYK2 (Tyr1054) Antibodies?

Commercial Phospho-TYK2 (Tyr1054) Antibodies share several key specifications while differing in certain aspects depending on the manufacturer. These antibodies are typically rabbit polyclonal IgGs produced against synthesized phosphopeptides derived from human TYK2 around the Tyr1054 residue . The concentration is generally standardized at 1 mg/mL, with recommended dilution ranges of 1:1000-2000 for Western blot applications . Storage requirements typically specify -20°C for long-term stability, with most formulations containing glycerol (20-50%) to prevent freeze-thaw damage . The antibodies are supplied in liquid format, often in PBS buffer with stabilizers like glycerol and preservatives such as sodium azide at 0.02% . Species reactivity commonly includes human and mouse, with some products also validated for rat samples . Applications validated for most products include Western blot, with some antibodies additionally tested for immunocytochemistry and immunofluorescence . The table below summarizes these specifications across products:

These specifications provide researchers with critical information for experimental planning and optimization when working with Phospho-TYK2 (Tyr1054) Antibodies.

What is the biological function of TYK2 and the significance of phosphorylation at Tyr1054?

TYK2 (Tyrosine Kinase 2) is a non-receptor tyrosine kinase belonging to the Janus kinase (JAK) family that plays crucial roles in cytokine signaling, particularly for type I interferons, IL-12, IL-23, and IL-10 family cytokines. TYK2 functions as a signal transducer that associates with the cytoplasmic domains of cytokine receptors and mediates signal transmission from the cell membrane to the nucleus . Following receptor engagement, TYK2 undergoes phosphorylation at multiple sites, with Tyr1054 representing a critical regulatory residue in the activation loop of its kinase domain. Phosphorylation at Tyr1054, especially when coupled with phosphorylation at the adjacent Tyr1055, induces a conformational change that activates the catalytic activity of TYK2 . This activation enables TYK2 to phosphorylate downstream substrates, particularly STAT (Signal Transducer and Activator of Transcription) proteins, which then dimerize, translocate to the nucleus, and initiate transcription of target genes . The biological significance of TYK2 extends to multiple physiological processes, including antiviral immunity, as noted in product descriptions: "TYK2 plays both structural and catalytic roles in type I interferon (IFN) signaling. As such, it may play a role in anti-viral immunity" . Additionally, mutations in TYK2 have been associated with primary immunodeficiency, specifically hyperimmunoglobulin E syndrome (HIES), highlighting its essential role in normal immune function .

How is TYK2 involved in disease states and what role does Tyr1054 phosphorylation play?

TYK2 has been implicated in various disease states, with phosphorylation at Tyr1054 serving as a critical regulatory event in pathological contexts. TYK2 mutations have been directly associated with hyperimmunoglobulin E syndrome (HIES), a primary immunodeficiency characterized by elevated serum immunoglobulin E levels, recurrent infections, and eczema . In this context, loss-of-function mutations can impair proper phosphorylation at regulatory sites including Tyr1054, disrupting downstream signaling cascades essential for antimicrobial immunity. Conversely, hyperactivation of TYK2 signaling, involving sustained phosphorylation at Tyr1054, has been linked to autoimmune and inflammatory conditions including psoriasis, inflammatory bowel disease, and systemic lupus erythematosus. The involvement of TYK2 in multiple immune-mediated disorders has made it an attractive therapeutic target, with several pharmaceutical companies developing TYK2 inhibitors that target its catalytic activity regulated by Tyr1054/1055 phosphorylation. The specific phosphorylation status of TYK2 at Tyr1054 can serve as both a biomarker for disease activity and a pharmacodynamic marker for therapeutic interventions targeting this pathway. Additionally, genetic studies have identified protective variants of TYK2 that alter phosphorylation potential at activation loop residues, providing natural models for therapeutic mimicry. Understanding the precise role of Tyr1054 phosphorylation in different disease contexts continues to inform both basic research and clinical development of targeted therapies.

What signaling pathways involve TYK2 phosphorylation at Tyr1054?

TYK2 phosphorylation at Tyr1054 serves as a critical regulatory event in multiple cytokine signaling pathways essential for immune regulation. The most well-characterized pathway involving TYK2 phosphorylation is the type I interferon (IFN-α/β) signaling cascade . In this pathway, binding of interferons to their heterodimeric receptor complex (IFNAR1/IFNAR2) induces conformational changes that activate receptor-associated TYK2 (bound to IFNAR1) and JAK1 (bound to IFNAR2), resulting in TYK2 phosphorylation at Tyr1054. Activated TYK2 then participates in phosphorylating STAT1 and STAT2, which form complexes with IRF9 (Interferon Regulatory Factor 9) to create the ISGF3 (Interferon-Stimulated Gene Factor 3) transcription factor complex. Beyond type I interferon signaling, TYK2 phosphorylation at Tyr1054 is involved in IL-12 signaling, where TYK2 associates with IL-12Rβ1 and contributes to STAT4 activation, promoting Th1 cell differentiation. Similarly, in IL-23 signaling, TYK2 phosphorylation mediates STAT3 activation, supporting Th17 cell development and function. The IL-10 family cytokines also utilize TYK2, where phosphorylation at Tyr1054 contributes to STAT3 activation in anti-inflammatory responses. These diverse signaling pathways highlight the central role of TYK2 Tyr1054 phosphorylation in coordinating both pro-inflammatory (IFN, IL-12, IL-23) and anti-inflammatory (IL-10) immune responses, explaining why dysregulation of TYK2 activity can contribute to various immunological disorders.

What are the optimal conditions for using Phospho-TYK2 (Tyr1054) Antibody in Western blot applications?

For optimal Western blot detection using Phospho-TYK2 (Tyr1054) Antibody, several critical parameters must be carefully controlled. Based on manufacturer recommendations, the antibody should be diluted to 1:1000-2000 in blocking buffer (typically 5% BSA in TBST) . Sample preparation requires particular attention to preserve phosphorylation status—cells should ideally be stimulated with appropriate cytokines such as type I interferons to induce TYK2 phosphorylation, and all lysis buffers must contain phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and β-glycerophosphate) to prevent dephosphorylation during processing . Cell lysates should be prepared freshly and kept cold throughout processing to maintain phospho-epitopes. For protein separation, 8% SDS-PAGE gels are recommended for optimal resolution in the high molecular weight range where TYK2 migrates (approximately 130-140 kDa). Transfer to PVDF membranes (rather than nitrocellulose) often yields superior results for phosphoprotein detection, with overnight transfer at low voltage (30V) in cold conditions to ensure complete transfer of large proteins. Blocking should be performed using 5% BSA in TBST rather than milk (which contains phosphoproteins that may interfere with detection) for 1-2 hours at room temperature. Primary antibody incubation is optimally performed overnight at 4°C with gentle agitation, followed by extensive washing (at least 3 × 10 minutes) with TBST. HRP-conjugated anti-rabbit secondary antibodies should be used at 1:5000-1:10000 dilution with incubation for 1 hour at room temperature. Enhanced chemiluminescence detection with longer exposure times may be necessary for optimal visualization of phospho-specific signals.

What controls should be included when using Phospho-TYK2 (Tyr1054) Antibody?

A robust experimental design using Phospho-TYK2 (Tyr1054) Antibody should incorporate multiple controls to ensure reliable and interpretable results. Positive controls should include lysates from cells treated with type I interferons (IFN-α/β), which robustly induce TYK2 phosphorylation at Tyr1054 . Negative controls should include unstimulated cells from the same cell line to establish baseline phosphorylation levels. A critical validation control involves treating duplicate samples with lambda phosphatase prior to immunoblotting; a genuine phospho-specific antibody will show abolished signal in phosphatase-treated samples while maintaining signal in untreated samples. For definitive validation, cells treated with JAK/TYK2 inhibitors prior to stimulation should show diminished phospho-TYK2 signal despite cytokine stimulation. Technical controls should include a primary antibody omission control to assess non-specific binding of secondary antibodies. For experiment normalization, membranes should be stripped and reprobed with antibodies detecting total TYK2 protein, allowing calculation of the phospho-to-total protein ratio that controls for expression level variations. Additionally, loading controls such as β-actin or GAPDH should be included to verify equal protein loading across samples. In immunocytochemistry applications, similar stimulation controls should be implemented, with the addition of peptide competition controls where the primary antibody is pre-incubated with the immunizing phosphopeptide, which should abolish specific staining. These comprehensive controls ensure that experimental observations made using Phospho-TYK2 (Tyr1054) Antibody are specific, reproducible, and biologically relevant.

How should samples be prepared to preserve TYK2 phosphorylation at Tyr1054?

Preserving TYK2 phosphorylation at Tyr1054 during sample preparation requires meticulous attention to multiple factors that can affect phosphoprotein stability. Cell stimulation protocols should be optimized for timing, with peak phosphorylation of TYK2 at Tyr1054 typically occurring 15-30 minutes after interferon treatment . Immediately following stimulation, cells should be rapidly lysed in ice-cold lysis buffer containing robust phosphatase inhibitor cocktails. Recommended buffer compositions include: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40 or 1% Triton X-100, 0.5% sodium deoxycholate, supplemented with phosphatase inhibitors (2 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM β-glycerophosphate, 1 mM EDTA) and protease inhibitors (1 mM PMSF, 5 μg/ml leupeptin, 5 μg/ml aprotinin) . Temperature control is critical—all steps should be performed at 4°C or on ice, including centrifugation to clear lysates, as even brief exposure to room temperature can result in significant dephosphorylation. Sample denaturation should be performed by adding Laemmli buffer and heating at 95°C for exactly 5 minutes; prolonged heating can damage phospho-epitopes. For tissue samples, snap-freezing in liquid nitrogen immediately after collection followed by pulverization while frozen and direct extraction in lysis buffer containing phosphatase inhibitors helps maintain phosphorylation status. Samples should be processed immediately whenever possible rather than stored, but if storage is necessary, lysates should be aliquoted in single-use volumes to avoid freeze-thaw cycles and stored at -80°C. These comprehensive measures help ensure that the phosphorylation status of TYK2 at Tyr1054 accurately reflects the biological state at the time of collection.

How can Phospho-TYK2 (Tyr1054) Antibody be used to study JAK-STAT pathway dynamics?

Phospho-TYK2 (Tyr1054) Antibody serves as a valuable tool for investigating the temporal and spatial dynamics of JAK-STAT pathway activation. To study signaling kinetics, researchers can design time-course experiments where cells are stimulated with cytokines for varying durations (typically ranging from 5 minutes to 24 hours), followed by quantitative Western blotting with Phospho-TYK2 (Tyr1054) Antibody . By plotting the phosphorylation intensity against time, researchers can determine the activation kinetics, peak phosphorylation time, and signal duration characteristics specific to different cytokines and cell types. For spatial dynamics analysis, immunocytochemistry or immunofluorescence applications using Phospho-TYK2 (Tyr1054) Antibody can reveal the subcellular localization of activated TYK2 . This approach can identify whether TYK2 phosphorylation occurs predominantly at membrane-proximal locations, cytoplasmic regions, or potentially translocates to different cellular compartments following activation. For pathway crosstalk studies, researchers can combine Phospho-TYK2 (Tyr1054) Antibody with antibodies against phosphorylated forms of other signaling molecules (like MAPKs, PI3K/Akt components) in multiplexed Western blots or immunofluorescence to determine how TYK2 activation correlates with other pathway activities. Signal integration studies can be designed by pre-treating cells with inhibitors of complementary pathways before cytokine stimulation, revealing how other signaling systems influence TYK2 phosphorylation at Tyr1054. Additionally, the antibody can be used in immunoprecipitation followed by mass spectrometry to identify proteins that specifically interact with phosphorylated TYK2, providing insights into signalosome composition and potential feedback regulators specific to the activated state of TYK2.

How can Phospho-TYK2 (Tyr1054) Antibody be used to validate TYK2 inhibitors?

Phospho-TYK2 (Tyr1054) Antibody provides a direct method for validating the efficacy of TYK2 inhibitors in preclinical and clinical research settings. For initial compound screening, the antibody can be used in cell-based assays where cells are pre-treated with candidate inhibitors prior to cytokine stimulation, followed by quantitative Western blotting to measure the reduction in TYK2 phosphorylation at Tyr1054 compared to uninhibited controls . This approach allows researchers to generate dose-response curves for inhibitor activity, determining IC50 values (concentration causing 50% inhibition of phosphorylation) that provide a quantitative measure of compound potency. For mechanistic studies, the antibody can distinguish between different classes of inhibitors—ATP-competitive inhibitors may not prevent Tyr1054 phosphorylation directly but block downstream substrate phosphorylation, while allosteric inhibitors may directly prevent Tyr1054 phosphorylation by stabilizing inactive conformations. Time-course experiments incorporating inhibitor washout can assess the durability of inhibition, revealing whether compounds achieve transient or sustained suppression of TYK2 phosphorylation. In ex vivo settings, the antibody can be used to measure target engagement in peripheral blood mononuclear cells isolated from patients receiving TYK2 inhibitors in clinical trials, providing a pharmacodynamic biomarker to correlate with clinical responses. High-content screening approaches using immunofluorescence with Phospho-TYK2 (Tyr1054) Antibody can evaluate inhibitor effects at the single-cell level, revealing population heterogeneity in drug responses. Additionally, combining the phospho-specific antibody with readouts of downstream pathway activation (phospho-STAT proteins) can determine whether reduced TYK2 phosphorylation correlates with functional suppression of the entire signaling cascade.

How can Phospho-TYK2 (Tyr1054) Antibody be used to investigate TYK2 mutations and variants?

Phospho-TYK2 (Tyr1054) Antibody provides a powerful tool for investigating how mutations and genetic variants affect TYK2 activation and function in both research and clinical contexts. For known disease-associated mutations, researchers can express wild-type and mutant TYK2 constructs in cell systems, then stimulate with appropriate cytokines and use the phospho-specific antibody to quantify differences in phosphorylation efficiency at Tyr1054 . This approach is particularly valuable for investigating mutations associated with primary immunodeficiencies like hyperimmunoglobulin E syndrome (HIES), where defective TYK2 phosphorylation may underlie pathogenesis . For structure-function studies, researchers can generate phosphorylation site mutants (Y1054F) and compare their signaling capabilities to wild-type TYK2, using the antibody to confirm the absence of phosphorylation in mutant constructs. When investigating naturally occurring genetic variants like single nucleotide polymorphisms (SNPs) associated with autoimmune disease risk, the antibody can assess whether these variants alter baseline or stimulus-induced phosphorylation at Tyr1054. For clinical applications, the antibody can be used in diagnostic immunoblotting to evaluate TYK2 phosphorylation status in patient-derived cells stimulated ex vivo, potentially identifying functional deficiencies even in cases where genetic testing is inconclusive. Additionally, CRISPR-engineered cell lines harboring specific TYK2 variants can be systematically evaluated for altered phosphorylation responses across multiple cytokine stimulations, creating comprehensive profiles of how genetic variation impacts TYK2 activation. This knowledge contributes to understanding personalized medicine approaches, where genetic profiles might predict responses to TYK2-targeting therapeutics.

What are common issues when using Phospho-TYK2 (Tyr1054) Antibody and how can they be resolved?

Researchers working with Phospho-TYK2 (Tyr1054) Antibody may encounter several common technical challenges that require systematic troubleshooting approaches. Weak or absent signal represents a frequent issue, potentially resulting from insufficient TYK2 phosphorylation in samples; this can be addressed by optimizing stimulation conditions with higher cytokine concentrations, verifying cytokine activity, and ensuring phosphatase inhibitors are fresh and used at appropriate concentrations . High background or non-specific bands may occur due to suboptimal blocking or antibody concentrations; resolving this requires titrating the antibody (starting at 1:2000 and adjusting as needed), extending blocking time to 2 hours with 5% BSA, and implementing more stringent washing procedures with increased TBST volume and number of washes . Signal variability between experiments may reflect inconsistencies in sample preparation or storage; standardizing lysate preparation protocols, avoiding freeze-thaw cycles, and using freshly prepared samples can improve reproducibility. Multiple bands on Western blots might represent degradation products, splice variants, or post-translational modifications; using protease inhibitors, freshly preparing samples, and running positive control lysates can help distinguish between these possibilities. For immunocytochemistry applications, high background fluorescence can result from inadequate blocking or fixation issues; optimization may include trying different fixatives (4% paraformaldehyde versus methanol), extending blocking time, and using specialized blocking reagents to reduce non-specific binding . Loss of phospho-signal over time in stored lysates indicates ongoing phosphatase activity despite inhibitors; this can be mitigated by adding additional phosphatase inhibitors immediately before gel loading and avoiding storage of diluted samples. By systematically addressing these common issues, researchers can obtain reliable and consistent results with Phospho-TYK2 (Tyr1054) Antibody.

How can the sensitivity of Phospho-TYK2 (Tyr1054) detection be improved?

Enhancing the sensitivity of Phospho-TYK2 (Tyr1054) detection requires optimization of multiple experimental parameters throughout the workflow. Beginning with sample preparation, researchers should implement phospho-enrichment strategies such as phosphotyrosine immunoprecipitation prior to immunoblotting, which can concentrate phosphorylated proteins from dilute samples . For Western blot applications, signal amplification systems such as SuperSignal West Femto substrate or similar high-sensitivity chemiluminescent reagents can significantly lower detection thresholds compared to standard ECL reagents. Optimizing protein loading is critical—for low abundance phosphoproteins like phospho-TYK2, increasing protein amounts to 50-80 μg per lane (rather than standard 20-30 μg) can improve detection without compromising resolution. Transfer efficiency can be enhanced by using PVDF membranes with 0.2 μm pore size (rather than 0.45 μm) and incorporating methanol-free transfer buffers specifically optimized for large proteins. Signal-to-noise ratio improvements can be achieved through extended blocking (overnight at 4°C) with specialized blocking reagents such as fish gelatin that provide lower background than conventional BSA or milk blockers. For immunocytochemistry applications, sensitivity can be dramatically improved using tyramide signal amplification (TSA) systems, which can enhance signal intensity 10-100 fold compared to conventional detection methods . Additionally, fluorescent Western blot systems with near-infrared detection offer superior sensitivity and broader dynamic range compared to chemiluminescence, allowing detection of low abundance phosphorylated species. For quantitative applications, implementing internal standard curves with recombinant phospho-proteins can calibrate detection sensitivity across experiments. These combined approaches can significantly enhance the detection limit for Phospho-TYK2 (Tyr1054), enabling analysis in challenging sample types or conditions with minimal phosphorylation.

How can specificity of Phospho-TYK2 (Tyr1054) Antibody be validated?

Validating the specificity of Phospho-TYK2 (Tyr1054) Antibody requires a multi-faceted approach that confirms selective recognition of the phosphorylated epitope in various experimental contexts. The gold-standard validation involves using genetically modified systems, specifically TYK2 knockout cells or CRISPR-engineered cells expressing TYK2 with tyrosine-to-phenylalanine mutations at position 1054 (Y1054F); absence of signal in these systems provides definitive confirmation of antibody specificity . Phosphatase treatment validation should be performed where duplicate samples are processed with and without lambda phosphatase treatment prior to immunoblotting; a truly phospho-specific antibody will show abolished signal in phosphatase-treated samples. Peptide competition assays provide another critical validation method, where pre-incubation of the antibody with excess phosphopeptide (used for immunization) should eliminate specific binding, while pre-incubation with non-phosphorylated peptide should not affect binding. Cross-reactivity assessment against other JAK family members (JAK1, JAK2, JAK3) with similar activation loop sequences can be performed using overexpression systems or specific stimulation conditions that preferentially activate individual JAK kinases. For applications in tissues or complex biological samples, immunoprecipitation followed by mass spectrometry can verify that the antibody enriches for peptides containing phosphorylated Tyr1054 in TYK2. Stimulus-response validation using dose-response and time-course experiments with known TYK2 activators (type I interferons) should demonstrate signal induction patterns consistent with established TYK2 biology . Finally, cross-validation using alternative phospho-specific antibodies from different commercial sources or using different detection techniques (such as ELISA versus Western blot) can provide additional confidence in antibody specificity. Implementing multiple validation approaches from this comprehensive strategy ensures robust and reliable results when using Phospho-TYK2 (Tyr1054) Antibody in research applications.