TXK Antibody

Basic Research Questions

• What is TXK and why are TXK antibodies important in immunological research?

  TXK (also known as RLK) is a non-receptor tyrosine kinase belonging to the Tec family that plays a critical role in T cell development, function, and differentiation of conventional T-cells and nonconventional NKT-cells . TXK antibodies are essential tools for studying T cell receptor signaling pathways and natural killer cell activation. Research shows that TXK expression is restricted to Th1/Th0 cells with IFN-γ producing potential, making TXK antibodies particularly valuable for investigating T helper cell differentiation . These antibodies enable researchers to track TXK expression patterns, protein interactions, and subcellular localization, providing insights into immune response regulation.

  TXK Function	Significance in Immune Response
  T cell receptor signaling	Mediates signal transduction following TCR activation
  IFN-γ production	Specifically regulates Th1 cytokine production
  Actin cytoskeleton regulation	Contributes to T cell migration and immunological synapse formation
  PLCG1 phosphorylation	Leads to calcium release and NFAT translocation to nucleus

• What are the primary experimental applications of TXK antibodies?

  TXK antibodies have multiple applications in immunological research:

  • Western Blotting (WB): Detects TXK protein expression levels and post-translational modifications. Studies have identified specific bands at approximately 58 and 65 kDa in lysates of Jurkat cells .

  • Immunoprecipitation (IP): Isolates TXK protein complexes to study protein-protein interactions, particularly with PARP1 and EF-1α in the IFN-γ promoter complex .

  • Immunofluorescence (IF)/Immunocytochemistry (ICC): Visualizes TXK subcellular localization in fixed cells, with protocols typically using PFA-fixed, Triton X-100 permeabilized cells .

  • Enzyme-Linked Immunosorbent Assay (ELISA): Provides quantitative measurement of TXK protein levels in solution .

  • Immunohistochemistry (IHC): Detects TXK expression in tissue samples, with optimized protocols using TE buffer pH 9.0 for antigen retrieval .

  Different antibodies have varying specificities for these applications. For example, Rabbit Polyclonal antibodies like ab233260 are suitable for WB, while others like ab262843 are optimized for ICC/IF applications .

• What is the expression pattern of TXK in immune cells?

  TXK exhibits a specific expression pattern primarily in T lymphocytes. Research using RT-PCR and antibody-based techniques has demonstrated that:

  • TXK is expressed in both peripheral blood CD4+ and CD8+ T cells

  • All Th1 cell clones and Th0 clones express TXK mRNA, whereas none of the Th2 clones express it

  • T cell lines including Jurkat and MOLT-4 express TXK

  • B cells, monocytes, and EBV-transformed B cells do not express TXK

  This restricted expression pattern makes TXK an excellent marker for distinguishing T cell subsets, particularly those with IFN-γ producing potential. Western blot analysis using specific TXK antibodies has successfully detected TXK in Jurkat human acute T cell leukemia cell line, showing specific bands at approximately 58 and 65 kDa .

• How should researchers select the appropriate TXK antibody for their experiments?

  Selection of the appropriate TXK antibody requires consideration of several experimental factors:

  • Application compatibility: Choose antibodies validated for your specific application (WB, IP, IF, IHC)

  • Epitope recognition: Consider antibodies targeting different regions of TXK based on your research question

    • Antibodies targeting aa 250 to C-terminus for detecting full-length protein

    • Antibodies targeting aa 50-150 for applications like ICC/IF

    • Antibodies recognizing aa 150-246 for functional studies

  • Host species: Select based on compatibility with other antibodies in multi-color applications

  • Clonality: Monoclonal antibodies offer higher specificity; polyclonal antibodies provide stronger signals

  • Validation data: Review data showing detection of endogenous TXK in relevant cell types

  Technical documentation recommends titrating antibodies in each testing system, with dilutions ranging from 1:10-1:100 for IF/ICC and 1:20-1:200 for IHC applications . Always verify reactivity with your species of interest, as some antibodies are specifically validated for human or mouse samples.

Advanced Research Questions

• How can TXK antibodies be used to investigate the role of TXK in T helper cell differentiation and IFN-γ regulation?

  TXK antibodies enable sophisticated experimental approaches to study its role in T helper differentiation:

  1. Chromatin Immunoprecipitation (ChIP): Using TXK antibodies for ChIP assays can identify genomic regions where TXK binds, particularly at the IFN-γ promoter where TXK forms a complex with PARP1 and EF-1α .

  2. Co-Immunoprecipitation (Co-IP): TXK antibodies can pull down TXK-associated proteins to identify interaction partners involved in T cell differentiation pathways .

  3. Phosphorylation Studies: Since TXK phosphorylates both PARP1 and EF-1α, antibodies detecting phosphorylated TXK (pTyr-420) can monitor TXK activation status during T cell differentiation .

  4. Knockdown/Inhibition Experiments: Combining TXK antibodies with antisense oligonucleotides targeting TXK confirms specific contribution of TXK to IFN-γ production. Research shows that antisense ODN specifically inhibits IFN-γ production in normal peripheral blood lymphocytes and antigen-specific Th1/Th0 clones without affecting IL-2 and IL-4 production .

  5. Time-Course Experiments: Using TXK antibodies in time-course experiments following T cell activation reveals the dynamics of TXK expression, phosphorylation, and nuclear translocation during Th1 cell differentiation.

• What are the technical considerations for optimizing Western blot detection of TXK?

  Optimizing Western blot detection of TXK requires careful attention to several technical aspects:

  1. Sample Preparation:

     • Use appropriate lysis buffers that preserve phosphorylation status when studying TXK activation

     • Include phosphatase inhibitors to prevent dephosphorylation of TXK

     • Consider subcellular fractionation to separate nuclear (55 kDa) and cytoplasmic/membrane-associated (~58-65 kDa) TXK forms

  2. Antibody Selection:

     • Choose antibodies validated specifically for Western blot applications

     • Consider epitope accessibility - some antibodies recognize specific domains of TXK

     • Human RLK/TXK Monoclonal Antibody (MAB6520) can detect specific bands at approximately 58 and 65 kDa in Jurkat cell lysates

  3. Running Conditions:

     • Use 8-10% SDS-PAGE gels for optimal resolution of TXK (calculated molecular weight ~61 kDa)

     • Include positive control lysates from cells known to express TXK (e.g., Jurkat cells)

     • Include negative control lysates from cells that don't express TXK (e.g., B cell lines)

  4. Detection Optimization:

     • Use reducing conditions to ensure consistent epitope exposure

     • Optimize primary antibody concentration (typically starting with 1 µg/mL for monoclonal antibodies)

     • Consider enhanced chemiluminescence (ECL) detection systems for sensitive detection

• How can in vitro kinase assays be designed using TXK antibodies to study its phosphorylation targets?

  In vitro kinase assays using TXK antibodies can investigate its phosphorylation activity and targets through the following methodological approach:

  1. TXK Immunoprecipitation:

     • Use TXK-specific antibodies (such as TXK Antibody B-2) to immunoprecipitate TXK from cell lysates

     • Include appropriate controls (isotype control antibodies for non-specific binding)

     • Consider using agarose-conjugated antibodies for efficient pull-down

  2. Kinase Reaction Setup:

     • Incubate recombinant TXK with potential substrate proteins (e.g., PARP1, EF-1α)

     • Use kinase buffer containing ATP (0.25 mM ATP in 40 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl₂, 3 mM MnCl₂, 1 mM DTT)

     • Include controls without ATP or with kinase inhibitors

  3. Detection of Phosphorylation:

     • Analyze reaction products by immunoblotting with antibodies against Txk, EF-1α, PARP1 and phosphotyrosine

     • Consider using radiolabeled ATP (γ-³²P-ATP) for higher sensitivity

  4. Validation of Results:

     • Perform mass spectrometry to identify the exact phosphorylation sites

     • Correlate in vitro findings with cellular experiments using TXK overexpression or knockdown

  Published research has demonstrated that TXK phosphorylates both PARP1 and EF-1α as part of an IFN-γ promoter-binding complex, and also phosphorylates key sites in LCP2 leading to the up-regulation of Th1-preferred cytokine IL-2 .

• What methodologies can be employed to study the subcellular localization of TXK using antibodies?

  Investigating TXK subcellular localization requires specific methodological approaches:

  1. Immunofluorescence/Immunocytochemistry:

     • Fix cells using PFA (typically 4%) and permeabilize with Triton X-100

     • Use TXK antibodies optimized for IF/ICC applications (such as ab262843)

     • Include markers for specific subcellular compartments (nuclear stains, membrane markers)

     • Analyze using confocal microscopy for precise localization

  2. Subcellular Fractionation followed by Western Blot:

     • Separate nuclear, cytoplasmic, and membrane fractions using differential centrifugation

     • Validate fraction purity using compartment-specific marker proteins

     • Perform Western blot using TXK antibodies on each fraction

     • Compare the distribution of the nuclear (~55 kDa) and cytoplasmic/membrane (~58-65 kDa) forms

  3. Live Cell Imaging:

     • For dynamic studies, use fluorescently labeled TXK antibody fragments

     • Track TXK translocation following T cell activation

     • Correlate with functional endpoints such as cytokine production

  Research has revealed that TXK exhibits dual localization patterns: a nuclear form that participates in transcriptional regulation of the IFN-γ gene and a cytoplasmic/membrane form recruited to the cell membrane following T cell receptor activation, where it undergoes phosphorylation at Tyr-420 .

• How can antisense oligonucleotide approaches be combined with TXK antibodies to study its functional role?

  Combining antisense oligonucleotide (ODN) approaches with TXK antibodies provides a powerful methodology:

  1. Antisense ODN Design and Validation:

     • Design antisense ODNs targeting TXK mRNA (e.g., sequence GAAACCTCATGGTAGCCC corresponding to the translation start site)

     • Include control sense ODNs (e.g., GGGCTACCATGAGGTTTC)

     • Optimize ODN concentration and incubation time for peripheral blood T cells or antigen-specific clones

  2. Verification of TXK Knockdown:

     • Use TXK antibodies to confirm reduced TXK protein expression

     • Quantify knockdown efficiency through analysis of cytoplasmic expression of TXK

  3. Functional Assays:

     • Stimulate cells with appropriate activators (e.g., PHA for peripheral blood T cells or antigen plus irradiated autologous PBMCs for antigen-specific T cell clones)

     • Measure cytokine production (particularly IFN-γ, IL-2, and IL-4) via ELISA or intracellular cytokine staining

     • Correlate cytokine production with TXK expression levels

  Research employing this approach has demonstrated that antisense ODN specifically inhibits cytoplasmic expression of TXK and selectively blocks IFN-γ production without affecting IL-2 or IL-4 production, confirming TXK's specific role in IFN-γ regulation .

• What approaches can researchers use to validate the specificity of TXK antibodies?

  Validation of TXK antibody specificity requires systematic approaches:

  1. Positive and Negative Controls:

     • Use cell lines with known TXK expression (Jurkat, MOLT-4) as positive controls

     • Use B cell lines (Raji, EBV-transformed B cells) as negative controls

     • Compare staining patterns across multiple cell types to confirm specificity

  2. Knockdown/Knockout Validation:

     • Use antisense oligonucleotides or siRNA to knockdown TXK expression

     • Compare antibody reactivity in knockdown versus control samples

     • For advanced validation, use CRISPR-based knockout systems

  3. Peptide Competition Assays:

     • Pre-incubate antibody with the immunizing peptide

     • Demonstrate loss of signal in the presence of the specific peptide

     • Show no effect with unrelated peptides

  4. Cross-Reactivity Assessment:

     • Test reactivity against related Tec family kinases (ITK, BTK)

     • Use recombinant proteins or overexpression systems

     • Consider epitope sequence alignment analysis

  5. Multiple Antibody Validation:

     • Compare results from multiple antibodies targeting different TXK epitopes

     • Confirm consistent expression patterns across antibodies

     • Correlate with mRNA expression data

• How can researchers troubleshoot non-specific binding issues with TXK antibodies?

  Troubleshooting non-specific binding requires systematic methodological approaches:

  1. Antibody Titration:

     • Perform careful titration experiments to determine optimal concentrations

     • For Western blots, test concentrations ranging from 0.5-5 μg/mL

     • For immunofluorescence, test dilutions from 1:10 to 1:200

  2. Blocking Optimization:

     • Test different blocking agents (BSA, non-fat milk, normal serum)

     • Optimize blocking time and temperature

     • Consider adding 0.1-0.5% Tween-20 in washing buffers

  3. Antigen Retrieval Methods:

     • For IHC, compare TE buffer pH 9.0 versus citrate buffer pH 6.0

     • Optimize retrieval time and temperature

     • Consider enzymatic versus heat-induced epitope retrieval

  4. Secondary Antibody Controls:

     • Include controls omitting primary antibody

     • Test for cross-reactivity with endogenous immunoglobulins

     • Consider using directly conjugated primary antibodies

  5. Sample Preparation:

     • Optimize fixation conditions for IF/IHC

     • For Western blot, ensure complete protein denaturation

     • Consider using fresh versus frozen samples

• What considerations are important when designing studies of TXK in T cell-mediated diseases?

  When investigating TXK in disease contexts, researchers should consider:

  1. Disease-Specific Expression Patterns:

     • TXK is expressed predominantly in Th1 cells in inflammatory diseases such as rheumatoid arthritis and Behcet's disease

     • Compare TXK expression levels between patient and healthy control samples

     • Correlate expression with disease activity markers

  2. Functional Studies:

     • Isolate T cells from patient samples for ex vivo analysis

     • Compare TXK-dependent signaling between patient and control cells

     • Use TXK antibodies to track activation status in disease states

  3. Therapeutic Targeting Potential:

     • Assess TXK as a biomarker for Th1-mediated pathologies

     • Evaluate TXK inhibition strategies using neutralizing antibodies

     • Correlate TXK activity with therapeutic responses

  4. Technical Considerations:

     • Optimize protocols for limited clinical samples

     • Consider using phospho-specific TXK antibodies to assess activation state

     • Develop standardized protocols for reproducible assessment across patient cohorts

  5. Integration with Other Markers:

     • Combine TXK analysis with assessment of downstream cytokines (IFN-γ)

     • Correlate with other T cell subset markers

     • Develop multiparameter flow cytometry panels including TXK