TXNRD2 Monoclonal Antibody

What is TXNRD2 and what is its biological significance in cellular redox homeostasis?

TXNRD2 (Thioredoxin Reductase 2) is a mitochondrial selenoprotein belonging to the pyridine nucleotide-disulfide oxidoreductase family and is a critical member of the thioredoxin (Trx) system. This enzyme is primarily involved in:

• Controlling reactive oxygen species (ROS) levels

• Regulating mitochondrial redox homeostasis

• Maintaining thioredoxin in a reduced state

• Participating in redox-regulated cell signaling pathways

Among the three mammalian thioredoxin reductase isozymes (TXNRD1, TXNRD2, and TXNRD3), TXNRD2 is specifically localized to mitochondria where it plays a crucial role in scavenging reactive oxygen species, making it essential for cellular defense against oxidative stress .

What are the optimal experimental conditions for using TXNRD2 monoclonal antibodies in Western blotting?

When performing Western blot analysis with TXNRD2 monoclonal antibodies, researchers should consider the following optimal conditions based on validated protocols:

For verification of antibody specificity, TXNRD2 knockout cell lines (such as Human TXNRD2 knockout HEK-293T cell line) are available as negative controls, with GAPDH (36 kDa) serving as a recommended loading control .

How can researchers validate the specificity of TXNRD2 monoclonal antibodies in their experimental systems?

Validating antibody specificity is crucial for reliable research outcomes. For TXNRD2 monoclonal antibodies, a multi-faceted approach is recommended:

Genetic Validation:

• Utilize TXNRD2 knockout cell lines as negative controls. For example, Human TXNRD2 knockout HEK-293T cell line (ab267267) has been used to demonstrate specificity of anti-TXNRD2 antibodies, where loss of signal confirmed specificity .

• Alternatively, establish TXNRD2-knockdown models using shRNA technology. Lentiviral shRNA transduction has been successfully used to create TXNRD2-knockdown H295R adrenocortical cell lines for investigating redox homeostasis .

Immunological Validation:

• Perform side-by-side comparisons with multiple antibodies targeting different epitopes of TXNRD2.

• Conduct peptide competition assays using the immunogenic peptide to block specific antibody binding.

Technical Validation:

• Compare reactivity across multiple cell lines with known TXNRD2 expression (validated examples include HEK293T, HepG2, K562, HeLa, and MCF-7 cells) .

• Utilize both reducing and non-reducing conditions in Western blotting to confirm expected molecular weight (54-57 kDa) .

• Include proper loading controls (e.g., GAPDH antibody) and perform densitometry for quantitative analysis .

Rigorous validation using these approaches ensures reliable detection of TXNRD2 and minimizes the risk of non-specific binding or false results.

What techniques beyond Western blotting can be effectively used with TXNRD2 monoclonal antibodies?

TXNRD2 monoclonal antibodies have demonstrated utility across multiple techniques beyond Western blotting:

Immunohistochemistry (IHC-P):

• Recommended dilutions range from 1:20 to 1:500, depending on the specific antibody .

• Antigen retrieval methods: TE buffer (pH 9.0) is suggested, though citrate buffer (pH 6.0) may serve as an alternative .

• Successfully applied to human liver tissue and various cancer tissue arrays .

• Protocol consideration: For paraffin-embedded sections, deparaffinization, rehydration, and appropriate blocking steps are essential.

Immunofluorescence (IF):

• Several TXNRD2 antibodies have been validated for IF applications, enabling subcellular localization studies .

• Co-staining with mitochondrial markers can confirm expected mitochondrial localization.

ELISA:

• Both direct and indirect ELISA formats have been validated with certain TXNRD2 antibodies .

• Recombinant TXNRD2 can serve as a positive control.

Flow Cytometry:

• For mitochondrial oxidative stress studies, MitoSOX Red has been used in conjunction with TXNRD2 assessment .

• Quantitative analysis can be performed using integrated median fluorescence intensity (iMFI), calculated by multiplying the frequency of events by the median fluorescence intensity .

Each technique requires specific optimization, and researchers should validate the chosen antibody for their particular application, as not all antibodies perform equally across all techniques.

What is the relationship between TXNRD2 dysfunction and disease pathogenesis?

TXNRD2 dysfunction has been implicated in several pathological conditions, with compelling evidence for its role in:

Familial Glucocorticoid Deficiency (FGD):

• A stop gain mutation (p.Y447X) in TXNRD2 has been identified in a consanguineous Kashmiri kindred with FGD .

• RT-PCR and Western blotting revealed complete absence of TXNRD2 in patients homozygous for this mutation .

• Functional studies using TXNRD2-knockdown H295R adrenocortical cells demonstrated impaired redox homeostasis, suggesting a mechanism for adrenal insufficiency .

Dilated Cardiomyopathy:

• Mutations in TXNRD2 have been identified as causative for dilated cardiomyopathy .

• Cardiac-specific deletion of Txnrd2 in mouse models has demonstrated the critical importance of this enzyme in myocardial protection after ischemia/reperfusion injury .

• Mitochondrial ROS, which TXNRD2 helps control, are capable of opening the mitochondrial permeability transition pore, a harmful event in cardiac ischemia/reperfusion .

Cancer Biology:

• TXNRD2 has been investigated as a potential biomarker in prostate cancer, with differential expression observed between tumor and stromal cells .

• Nanotechnology-based detection methods have been developed to distinguish prostate cancer-associated stroma from benign prostatic hyperplasia based on thioredoxin-interacting protein partners .

This growing body of evidence highlights the importance of TXNRD2 in maintaining cellular redox balance and suggests that therapeutic approaches targeting TXNRD2 function might be valuable in managing these conditions.

How can researchers optimize TXNRD2 detection in challenging tissue samples?

Detecting TXNRD2 in challenging tissue samples requires specialized approaches:

Tissue-Specific Optimization Strategies:

• Fixation Protocol Refinement:

  • For formalin-fixed paraffin-embedded (FFPE) tissues, optimal fixation time (typically 24 hours) is critical.

  • Over-fixation can mask epitopes while under-fixation may compromise tissue morphology.

• Enhanced Antigen Retrieval Methods:

  • For TXNRD2 detection, TE buffer (pH 9.0) is generally recommended, but optimization is tissue-dependent .

  • Consider testing multiple retrieval methods: heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0), EDTA buffer (pH 8.0), or enzymatic retrieval with proteinase K.

  • Extended retrieval times (15-20 minutes) may improve detection in dense tissues.

• Signal Amplification Techniques:

  • Tyramide signal amplification (TSA) can significantly enhance sensitivity.

  • Polymer-based detection systems generally provide better results than avidin-biotin methods for low-abundance proteins like TXNRD2.

• Background Reduction Strategies:

  • Pre-incubation with 3% hydrogen peroxide effectively blocks endogenous peroxidase.

  • For tissues with high autofluorescence, Sudan Black B treatment (0.1% in 70% ethanol) can reduce background.

  • Tissues with high biotin content benefit from avidin-biotin blocking steps.

• Controls and Validation:

  • Include TXNRD2 knockout tissues or cells as negative controls .

  • Use tissues with known high TXNRD2 expression (such as liver) as positive controls .

These approaches should be systematically evaluated and combined as needed to achieve optimal TXNRD2 detection in challenging samples.

What are the differences between various commercially available TXNRD2 monoclonal antibodies?

Commercially available TXNRD2 monoclonal antibodies differ in several key aspects that may influence their performance in specific applications:

Key Considerations for Selection:

• Application Compatibility:

  • For Western blotting, the Abcam and AbClonal antibodies have shown high specificity with validated knockout controls .

  • For IHC applications, dilution requirements vary significantly (1:20 for NovoPro vs. 1:50-1:500 for Proteintech) .

• Species Cross-Reactivity:

  • While most antibodies react with human TXNRD2, those requiring mouse or rat reactivity should select antibodies specifically validated in these species .

• Epitope Consideration:

  • Antibodies targeting different epitopes may perform differently depending on protein conformation, post-translational modifications, or protein interactions.

  • The AbClonal antibody targets the N-terminal region (amino acids 1-100) , which may be advantageous for detecting specific isoforms.

• Validation Rigor:

  • The Abcam antibody has been validated using TXNRD2 knockout cell lines, providing strong evidence of specificity .

  • Publication citations can indicate successful use in peer-reviewed research.

These differences underscore the importance of selecting the appropriate antibody based on the specific experimental requirements and validation needs.

How does TXNRD2 function in the mitochondrial redox regulation system?

TXNRD2 plays a central role in mitochondrial redox regulation through several interconnected mechanisms:

Electron Transfer Mechanism:

TXNRD2 functions as a homodimeric flavoenzyme containing FAD and a selenocysteine residue at its C-terminal active site. It catalyzes the NADPH-dependent reduction of thioredoxin (Trx2) in mitochondria following this reaction cascade:

$\text{NADPH} + \text{H}^+ + \text{Trx2-S}_2 \xrightarrow{\text{TXNRD2}} \text{NADP}^+ + \text{Trx2-(SH)}_2$

The reduced thioredoxin then directly reduces various substrates, including peroxiredoxins, which detoxify hydrogen peroxide and other reactive oxygen species .

Mitochondrial ROS Management:

• TXNRD2 is essential for scavenging mitochondrial reactive oxygen species, particularly those generated during oxidative phosphorylation .

• It maintains mitochondrial peroxiredoxin 3 (Prx3) in its reduced, active state, enabling continuous detoxification of H₂O₂ .

• Studies using TXNRD2-knockdown cells have demonstrated increased mitochondrial ROS levels, confirming its critical role in ROS management .

Redox Signaling Integration:

• Beyond direct ROS detoxification, TXNRD2 participates in redox-regulated cell signaling pathways .

• It influences mitochondrial membrane potential and can affect mitochondrial permeability transition pore opening, which is particularly relevant in cardiac ischemia/reperfusion injury .

• The thioredoxin system regulated by TXNRD2 modulates the activity of various redox-sensitive transcription factors and enzymes.

Pathophysiological Significance:

• TXNRD2 dysfunction leads to impaired redox homeostasis, as demonstrated in studies of familial glucocorticoid deficiency .

• In cardiac tissue, TXNRD2 is essential for early postischemic myocardial protection, with its absence leading to increased susceptibility to oxidative damage .

• Mitochondrial thioredoxin reductase activity is particularly crucial in tissues with high metabolic rates and oxygen consumption.

This multifaceted role highlights TXNRD2 as a critical component of the cellular defense system against oxidative stress, particularly within mitochondria where the majority of cellular ROS are generated.

What are the most effective protocols for troubleshooting non-specific binding of TXNRD2 antibodies?

When facing non-specific binding issues with TXNRD2 antibodies, researchers can implement the following systematic troubleshooting approach:

Diagnostic Steps:

• Identify Pattern of Non-specificity:

  • Multiple bands in Western blotting

  • Unexpected cellular localization in IHC/IF

  • Signal in negative control samples

For Western Blotting:

• Antibody Optimization:

  • Perform dilution series (1:1000-1:6000) to identify optimal concentration .

  • Test multiple primary antibody incubation times and temperatures (overnight at 4°C vs. 1-2 hours at room temperature).

• Blocking Improvements:

  • Test alternative blocking solutions (5% BSA vs. 3-5% non-fat milk in TBST) .

  • Extend blocking time to 2 hours at room temperature.

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions.

• Sample Preparation Refinement:

  • Ensure complete protein denaturation (heat samples at 95°C for 5 minutes).

  • Test both reducing and non-reducing conditions, as some epitopes may be sensitive to reduction .

  • Include protease inhibitors in lysis buffer to prevent degradation products.

• Washing Optimization:

  • Increase wash duration and number of washes (5 × 5 minutes with TBST).

  • Add 0.05-0.1% SDS to washing buffer for stubborn non-specific binding.

For Immunohistochemistry/Immunofluorescence:

• Antigen Retrieval Enhancement:

  • Compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0) .

  • Optimize retrieval time and temperature.

• Antibody Diluent Modification:

  • Add 1-5% normal serum from the same species as the secondary antibody.

  • Include 0.1-0.3% Triton X-100 for better tissue penetration.

• Validation Controls:

  • Use TXNRD2 knockout tissues/cells as definitive negative controls .

  • Pre-absorb antibody with recombinant TXNRD2 protein to confirm specificity.

Verification Approach:

After implementing troubleshooting measures, confirm improved specificity by:

• Comparing results with at least one alternative TXNRD2 antibody from a different supplier

• Testing antibody performance in cells with known TXNRD2 expression levels

• Using protein loading controls (e.g., GAPDH) to normalize expression and confirm equal loading

This methodical approach addresses the most common sources of non-specific binding while providing validation strategies to confirm authentic TXNRD2 detection.

How can researchers effectively design experiments to study TXNRD2 function in disease models?

Designing robust experiments to study TXNRD2 function in disease models requires a multifaceted approach:

Knockout/Knockdown Models:

• Generate tissue-specific TXNRD2 knockout mouse models using Cre-lox technology for organ-specific studies (particularly valuable for cardiac research) .

• Establish TXNRD2-knockdown cell lines using lentiviral shRNA transduction as demonstrated with H295R adrenocortical cells .

• Utilize CRISPR-Cas9 gene editing for precise mutation introduction mimicking disease-associated variants.

Rescue Experiments:

• Reintroduce wild-type or mutant TXNRD2 into knockout models to confirm phenotype specificity.

• Use selenocysteine-to-cysteine mutants to assess the importance of selenocysteine in TXNRD2 function.

Redox Status Assessment:

• Measure ROS levels using fluorescent probes specific for mitochondrial oxidants (MitoSOX Red) .

• Quantify reduced/oxidized glutathione ratios as indicators of cellular redox state.

• Assess thioredoxin redox state using redox Western blotting with non-reducing conditions.

Mitochondrial Function Analysis:

• Evaluate mitochondrial membrane potential using JC-1 or TMRM dyes.

• Measure oxygen consumption rate (OCR) using Seahorse XF analyzers.

• Assess mitochondrial morphology through electron microscopy and confocal imaging.

For Cardiomyopathy Research:

• Implement ischemia/reperfusion injury models to evaluate TXNRD2's role in cardioprotection .

• Use cardiac stress tests (pressure overload, drug-induced cardiotoxicity) to assess the impact of TXNRD2 deficiency.

For Adrenal Disorders:

• Develop cellular models mimicking familial glucocorticoid deficiency through TXNRD2 mutation .

• Assess steroidogenic capacity in TXNRD2-deficient adrenocortical cells.

For Cancer Studies:

• Compare TXNRD2 expression and function between tumor and adjacent normal tissues .

• Evaluate the impact of TXNRD2 inhibition on cancer cell survival and chemosensitivity.

Biomarker Development:

• Assess TXNRD2 protein levels in patient samples using validated antibodies .

• Develop activity assays to measure TXNRD2 enzymatic function in clinical specimens.

Therapeutic Targeting:

• Screen for compounds that can restore TXNRD2 function in disease models.

• Evaluate antioxidant therapies as compensatory approaches in TXNRD2-deficient states.