TYK2 Antibody, FITC conjugated

What is TYK2 and what are its key cellular functions?

TYK2 is a non-receptor tyrosine protein kinase belonging to the Janus kinase (JAK) family. It associates with the cytoplasmic domain of type I and type II cytokine receptors and propagates cytokine signals by phosphorylating receptor subunits. TYK2 is a critical component of both type I and type III interferon signaling pathways and plays important roles in:

• Initiating type I interferon signaling

• Phosphorylating the interferon-alpha/beta receptor alpha chain

• Mediating IL-12, IL-23, IL-10, and IFN-α/β signaling

• Anti-viral immunity

• Regulating autoimmune responses

Cellular studies have shown that TYK2 deficiency leads to impaired but not abolished cellular responses to multiple cytokines, demonstrating its central role in immune signaling networks .

What are the primary applications for FITC-conjugated TYK2 antibodies?

FITC-conjugated TYK2 antibodies are valuable tools for fluorescent detection of TYK2 protein. Their primary applications include:

• Flow cytometry analysis of TYK2 expression in different cell populations

• Immunofluorescence microscopy to visualize subcellular localization

• Western blotting (WB) for protein detection

• Immunoprecipitation (IP) to isolate TYK2 protein complexes

• ELISA-based detection systems

The fluorescent conjugation allows direct visualization without the need for secondary antibodies, making these reagents particularly useful for multicolor flow cytometry and fluorescence microscopy applications .

What is the specificity and cross-reactivity of commercially available TYK2-FITC antibodies?

Available TYK2-FITC antibodies show different specificity profiles:

When selecting an antibody, researchers should consider:

• The target species being studied

• The specific region of TYK2 being recognized

• Potential cross-reactivity with related JAK family members

• Validation in knockout or knockdown models

Detailed validation data should be requested from manufacturers to confirm specificity for your particular experimental system .

What are the optimal storage and handling conditions for TYK2-FITC antibodies?

To maintain antibody functionality and fluorescence signal:

• Store at -20°C for long-term storage

• Aliquot into multiple vials to avoid repeated freeze-thaw cycles

• Protect from light to preserve FITC fluorescence

• Most FITC-conjugated antibodies are supplied in storage buffers containing stabilizers

  • For example, the Bioss antibody is provided in "Aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol"

• Check manufacturer's recommendations for specific lot expiration dates and stability information

Proper storage and handling significantly impact experimental reproducibility and signal strength .

What are the recommended working dilutions for different applications of TYK2-FITC antibodies?

Optimal working dilutions vary by application:

*Based on typical FITC-conjugated antibody dilutions, specific titrations should be performed.

Always perform antibody titrations in your specific experimental system to determine optimal concentrations that maximize signal-to-noise ratios .

How can TYK2-FITC antibodies be used to study TYK2 activation in different immune cell populations?

TYK2-FITC antibodies are valuable tools for analyzing cell-type specific TYK2 expression and activation patterns:

Methodology:

• Isolate primary immune cells (macrophages, NK cells, CD8+ T cells)

• Treat with cytokines that activate TYK2 (IFN-α, IL-12, IL-23)

• Fix and permeabilize cells for intracellular staining

• Co-stain with TYK2-FITC antibody and cell surface markers

• Analyze by flow cytometry or imaging cytometry

Research has demonstrated cell-type-specific effects of TYK2 in different immune populations. For example, studies of splenic immune cells showed that TYK2 had pronounced, cell-type-specific effects on interferon-stimulated gene (ISG) signatures, with macrophages showing a stronger TYK2-dependent ISG signature compared to NK and CD8+ T cells .

To study TYK2 activation specifically, combine TYK2-FITC antibodies with phospho-specific antibodies against downstream STAT proteins (STAT1, STAT3, STAT5) to simultaneously detect expression and activation of the signaling pathway .

What protocols can be used to validate TYK2-FITC antibody specificity in knockout or inhibitor-treated samples?

Validation Protocol:

• Genetic Validation:

  • Use TYK2-deficient (Tyk2-/-) cells or tissues

  • Use TYK2 kinase-inactive (Tyk2K923E) samples

  • Compare staining patterns with wild-type samples

• Pharmacological Validation:

  • Treat cells with TYK2 inhibitors (e.g., deucravacitinib or other JH2 pseudokinase domain inhibitors)

  • Compare staining patterns before and after inhibitor treatment

• Sibling Specificity Control:

  • Include staining with isotype-matched FITC-conjugated antibodies

  • Test with non-peptide immunogen-blocked antibody

• Epitope Masking:

  • Use intrabodies like HJ8.5 that mask the epitope region (if the antibody targets a known epitope)

  • Test specificity in controlled expression systems using Y29F mutants if targeting this region

Research has demonstrated the importance of proper validation, as exemplified in phosphorylation studies where antibody specificity was confirmed using both Y29F mutations and intrabody masking approaches .

How can researchers effectively use TYK2-FITC antibodies in flow cytometry for detecting activation of TYK2-dependent pathways?

Protocol for Flow Cytometric Analysis of TYK2-Dependent Signaling:

• Sample Preparation:

  • Isolate cells of interest (e.g., PBMCs, cultured cell lines)

  • Stimulate with appropriate cytokines (IFN-α, IL-12, IL-23)

  • Include unstimulated controls and appropriate inhibitor controls

• Staining Procedure:

  • Fix cells with 1-4% paraformaldehyde (10-15 minutes)

  • Permeabilize with methanol or commercial permeabilization buffers

  • Block with appropriate blocking buffer (containing serum)

  • Stain with TYK2-FITC antibody plus lineage markers

  • For dual detection of phosphorylated downstream targets:

    • Co-stain with PE or APC-conjugated phospho-STAT antibodies

• Analysis Strategy:

  • Gate on viable cells and specific cell populations

  • Compare TYK2 expression levels across cell types

  • Analyze correlation between TYK2 expression and pSTAT levels

This approach has been successfully used in studies examining IFN-α-induced phosphorylation of STAT5 in CD3+ T cells in human whole blood, where researchers could detect both TYK2 expression and downstream signaling activation .

What are the methodological considerations for using TYK2-FITC antibodies to study the effects of novel TYK2 inhibitors?

Experimental Design for Inhibitor Studies:

• Dose-Response Analysis:

  • Treat cells with serial dilutions of TYK2 inhibitor

  • Include positive controls (known TYK2 inhibitors like deucravacitinib)

  • Include negative controls (inactive analog compounds)

• Time-Course Studies:

  • Evaluate inhibitor effects at multiple time points

  • Pre-incubation periods of 1-2 hours are typically used before cytokine stimulation

  • Assess both immediate (15-30 minutes) and delayed (4-24 hours) responses

• Readout Methods:

  • Direct visualization of TYK2 localization with TYK2-FITC antibody

  • Co-staining for downstream phospho-proteins

  • Quantification of inhibition by flow cytometry median fluorescence intensity (MFI)

• Validation in Multiple Cell Types:

  • Test inhibitor effects in T cells, NK cells, and macrophages

  • Compare inhibitor potency across species (human, mouse, rat)

Research has shown that TYK2 inhibitors targeting the pseudokinase (JH2) domain have different effects compared to kinase domain inhibitors. Flow cytometry with appropriate antibodies can quantify these differences, as demonstrated in studies where TYK2 JH2 ligands suppressed IFN-α-induced phosphorylation of STAT5 in CD3+ T cells in a dose-dependent manner .

How can TYK2-FITC antibodies be used alongside RNA sequencing to correlate protein expression with transcriptional responses?

Integrated Protein-Transcriptome Analysis Protocol:

• Cell Isolation and Splitting:

  • Isolate target cells (e.g., primary immune cells or cell lines)

  • Split sample for parallel processing:

    • Flow cytometry (protein analysis)

    • RNA extraction (transcriptome analysis)

• Treatment Conditions:

  • Apply identical treatments to both sample sets:

    • Cytokine stimulation (IFN-α, IL-12, IL-23)

    • Inhibitor treatments

    • Time course collection

• Flow Cytometry Analysis:

  • Stain with TYK2-FITC antibody and cell surface markers

  • Quantify TYK2 protein expression levels by MFI

  • Sort cells based on TYK2 expression levels if desired

• RNA-Seq Analysis:

  • Extract RNA from parallel samples

  • Perform RNA sequencing

  • Analyze interferon-stimulated gene (ISG) signatures

• Data Integration:

  • Correlate TYK2 protein levels with ISG expression patterns

  • Compare effects across cell types and treatments

This approach has revealed that TYK2 has pronounced, cell-type-specific effects on the ISG signature, with macrophages showing almost exclusively ISG dependence on TYK2, while NK and CD8+ T cells show more diverse gene expression patterns dependent on TYK2 .

What techniques can be used to study the role of TYK2 in tonic interferon signaling using TYK2-FITC antibodies?

Methodological Approach for Studying Tonic Signaling:

• Baseline TYK2 Expression Analysis:

  • Compare TYK2-FITC staining intensity across untreated cell types

  • Quantify nuclear vs. cytoplasmic localization

  • Compare wild-type vs. TYK2-deficient or kinase-inactive controls

• Depletion Studies:

  • Use anti-interferon antibodies to neutralize endogenous interferons

  • Monitor changes in TYK2 expression and localization

• Combined Flow-qPCR Analysis:

  • Sort cells based on TYK2-FITC expression levels

  • Perform qPCR for tonic ISG expression in sorted populations

  • Compare ISG patterns in high vs. low TYK2-expressing cells

• Chromatin Accessibility Correlation:

  • Sort cells based on TYK2-FITC expression

  • Perform ATAC-seq on sorted populations

  • Correlate chromatin accessibility at ISG promoters with TYK2 levels

Research has shown that TYK2 maintains cell-type-specific patterns of tonically expressed ISGs, with macrophages, NK cells, and CD8+ T cells showing distinct tonic ISG signatures that are differentially dependent on TYK2 kinase activity .

How can TYK2-FITC antibodies be used to investigate TYK2's role in tau phosphorylation and neurodegenerative disorders?

Neurodegenerative Research Protocol:

• Co-localization Studies:

  • Stain neuronal cells with TYK2-FITC antibody

  • Co-stain with antibodies against tau protein

  • Analyze co-localization using confocal microscopy

• Phosphorylation Analysis:

  • Stimulate neurons with cytokines known to activate TYK2

  • Immunoprecipitate tau using anti-tau antibodies

  • Probe for phosphorylated tau (particularly at Y29) by Western blot

  • Compare results in presence/absence of TYK2 inhibitors

• Aggregation Assays:

  • Express fluorescently-tagged tau (tau441-P301S-YFP) in cells

  • Monitor tau aggregation with/without TYK2 overexpression

  • Treat with TYK2 inhibitors (e.g., deucravacitinib)

  • Quantify changes in aggregate formation

• Primary Neuron Studies:

  • Isolate primary neurons from wild-type and TYK2-deficient mice

  • Stain with TYK2-FITC to confirm genotype

  • Compare tau phosphorylation and aggregation

Recent research has revealed that TYK2 phosphorylates tau protein at tyrosine 29 (Tyr29) residue (Y29) and stabilizes its levels in human cells and cultured mouse primary neurons. TYK2 enhances the aggregation of pathogenic tau (tau441-P301S), except when Y29 is mutated to phenylalanine. These findings suggest TYK2 may be a novel therapeutic target for tauopathies .

What controls should be included when using TYK2-FITC antibodies for immunofluorescence microscopy?

Essential Controls for Immunofluorescence:

• Antibody Specificity Controls:

  • TYK2-deficient cells or tissues

  • TYK2 siRNA-treated samples

  • Pre-adsorption with immunizing peptide

  • Isotype-matched FITC-conjugated control antibody

• Technical Controls:

  • Single-color controls for spectral compensation

  • Unstained samples for autofluorescence determination

  • Secondary-only controls (if using additional unconjugated antibodies)

  • FITC-conjugated irrelevant antibody (same concentration)

• Biological Controls:

  • TYK2 inhibitor-treated samples

  • Cytokine-stimulated vs. unstimulated cells

  • Cells expressing known high vs. low levels of TYK2

• Co-localization Controls:

  • Positive co-localization control (proteins known to interact)

  • Negative co-localization control (proteins in different subcellular compartments)

Properly controlled immunofluorescence can reveal the subcellular distribution of TYK2, which has been reported in multiple compartments including cytoplasm, nucleus, cytoskeleton, and plasma membrane .

What methodological approaches can be used to study cell-type specific effects of TYK2 using FITC-conjugated antibodies?

Cell-Type Specific Analysis Protocol:

• Multi-Parameter Flow Cytometry:

  • Isolate mixed cell populations (e.g., PBMCs, splenocytes)

  • Stain with TYK2-FITC plus lineage markers:

    • Macrophages: CD11b, F4/80

    • NK cells: CD56, CD16

    • T cells: CD3, CD4, CD8

    • B cells: CD19, CD20

  • Analyze TYK2 expression levels across populations

• Imaging Flow Cytometry:

  • Combine flow cytometry with microscopy

  • Analyze subcellular localization of TYK2-FITC in different cell types

  • Quantify nuclear translocation upon cytokine stimulation

• Cell Sorting and Functional Assays:

  • Sort cells based on TYK2-FITC expression level

  • Perform functional assays on sorted populations:

    • Cytokine production

    • Proliferation

    • Cytotoxicity (NK cells)

• Ex Vivo Tissue Analysis:

  • Prepare tissue sections from relevant organs

  • Stain with TYK2-FITC and lineage markers

  • Analyze tissue-specific expression patterns

This approach aligns with research findings showing distinct TYK2-dependent transcriptomic signatures in different immune cell types, where the impact of TYK2 deficiency or kinase inactivation varies significantly between macrophages, NK cells, and T cells .

How can researchers use TYK2-FITC antibodies to study the effects of TYK2 inhibitors on interferon-repressed genes?

IFN-Repressed Gene Analysis Protocol:

• Cell Preparation:

  • Culture target cells (primary immune cells or cell lines)

  • Pre-treat with TYK2 inhibitors or vehicle control

  • Stimulate with IFN-α/β (short-term and long-term timepoints)

• Flow Cytometry Analysis:

  • Stain with TYK2-FITC antibody to verify target engagement

  • Co-stain for markers of cell activation

  • Analyze expression patterns by flow cytometry

• Cell Sorting:

  • Sort cells based on TYK2-FITC staining intensity

  • Isolate RNA from sorted populations

  • Perform qPCR or RNA-seq for IFN-repressed genes

• Inhibitor Titration:

  • Test multiple inhibitor concentrations

  • Generate dose-response curves

  • Correlate inhibitor concentration with:

    • TYK2-FITC staining pattern changes

    • Expression of IFN-repressed genes

Recent research has identified a previously unrecognized role for TYK2 in regulating interferon-repressed genes (IRepG). Notably, TYK2 is indispensable for both early and late IFN-β-repressed gene profiles. Unlike the partial requirement for TYK2 in ISG induction, complete TYK2 activity is required for proper repression of IRepG, revealing a novel aspect of TYK2 biology with potential therapeutic implications .