TXN2 Antibody

What is TXN2 and what cellular functions does it regulate?

TXN2 (Thioredoxin 2) is a mitochondrial member of the thioredoxin family, a group of small multifunctional redox-active proteins. The protein plays important roles in regulating mitochondrial membrane potential and providing protection against oxidant-induced apoptosis . TXN2 is critical for maintaining cellular redox homeostasis and protecting cells from oxidative stress-induced damage. Research indicates that overexpression of TXN2 in human 143B osteosarcoma cells leads to increased cell viability under oxidative stress conditions, while chicken DT40 B cells deficient for TXN2 display increased intracellular reactive oxygen species (ROS) and apoptosis . In mice, complete loss of the TXN2 gene results in embryonic lethality at E10.5-12.5, demonstrating its essential role in development .

What is the observed molecular weight of TXN2 in Western blot applications?

When detecting TXN2 using Western blot analysis, researchers should expect to observe a specific band at approximately 13-14 kDa . It's worth noting that the calculated molecular weight based on amino acid sequence is around 18 kDa , but the actual observed weight in SDS-PAGE is slightly lower. This discrepancy between calculated and observed molecular weights is common for many proteins due to factors such as post-translational modifications, protein folding characteristics, and the reducing conditions of SDS-PAGE. When performing Western blot, it's advisable to include positive control samples with known TXN2 expression, such as human HEL, THP-1, K562, or 293T cell lysates, as these have demonstrated clear TXN2 bands in validation studies .

What applications are TXN2 antibodies validated for in research settings?

TXN2 antibodies have been validated for multiple research applications depending on the specific antibody product. Comprehensive validation typically includes Western blot (WB), immunofluorescence (IF), immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemistry (IHC) . For example, the Boster Bio Anti-Thioredoxin 2/TXN2 Antibody (A04586-1) has been validated for ELISA, Flow Cytometry, IF, IHC, ICC, and WB applications . The recommended dilution ranges vary by application, with Western blot typically using 1:500-1:2000 dilutions and IF/ICC applications requiring more concentrated antibody solutions at 1:10-1:200, depending on the specific antibody product . Researchers should always refer to the datasheet of their specific antibody for optimal application-specific dilutions.

What species reactivity is commonly available for TXN2 antibodies?

Commercial TXN2 antibodies exhibit varying cross-reactivity profiles across species. Based on the provided search results, many TXN2 antibodies react with human and mouse samples . Some antibodies offer broader reactivity, such as the Boster Bio Anti-Thioredoxin 2/TXN2 Antibody (A04586-1), which has been validated to react with human, mouse, and rat specimens . This cross-reactivity makes these antibodies versatile tools for comparative studies across different model organisms. When selecting a TXN2 antibody for your research, it's essential to verify that the antibody has been validated for your species of interest to ensure reliable results. Always check the manufacturer's documentation for specific information about species reactivity testing methodology.

What are the optimal fixation and permeabilization techniques for TXN2 immunofluorescence studies?

For successful immunofluorescence detection of TXN2, optimized fixation and permeabilization protocols are essential since TXN2 is a mitochondrial protein. Based on published methodologies, the following approach has proven effective: For cultured cells, fixation with 4% paraformaldehyde followed by permeabilization with an appropriate permeabilization buffer is recommended . For tissue sections, heat-mediated antigen retrieval using citrate buffer (pH 6.0) for approximately 20 minutes has shown good results . When working with cell lines like NIH/3T3 or U2OS, standard IF protocols using 1:100 dilution of TXN2 antibody demonstrate clear mitochondrial staining patterns . The choice of secondary antibodies should match the host species of the primary antibody, with conjugated fluorophores selected based on your imaging system's capabilities. For optimal visualization, counterstaining with DAPI for nuclear visualization helps contextualize the mitochondrial localization of TXN2 .

What are the recommended controls when validating TXN2 antibody specificity?

When validating the specificity of TXN2 antibodies, multiple control strategies should be implemented to ensure reliable results. Positive controls should include cell lines or tissues with known TXN2 expression. Based on validation data, human HEL, THP-1, K562, and 293T cell lines show clear TXN2 expression and serve as excellent positive controls for Western blot applications . For negative controls, consider using: (1) Primary antibody omission controls; (2) Isotype controls using non-specific IgG from the same host species; (3) Peptide competition assays where the antibody is pre-incubated with excess antigen peptide before application; and (4) If available, TXN2 knockout or knockdown samples provide the most stringent negative controls. For validating specificity between closely related proteins, such as TXN1 and TXN2, Western blot analysis looking at the molecular weight differences (TXN2 at ~13-14 kDa) and subcellular fractionation showing mitochondrial localization for TXN2 can provide additional evidence of specificity .

How should sample preparation differ when detecting TXN2 in different cell types?

Sample preparation protocols for TXN2 detection should be optimized based on the cell type and experimental application. For adherent cell lines (e.g., 293T, U2OS), standard lysis with RIPA buffer supplemented with protease inhibitors is generally sufficient for Western blot applications . For suspension cells (e.g., THP-1, K562), gentle centrifugation followed by similar lysis procedures works well . When working with tissue samples, mechanical homogenization in appropriate lysis buffer prior to clarification is recommended, as demonstrated with heart and kidney tissue lysates from both rat and mouse models . For immunofluorescence studies, different cell types may require adjusted fixation times: For U2OS cells, standard fixation protocols with 4% paraformaldehyde yield clear mitochondrial staining patterns , while THP-1 cells for flow cytometry benefit from 4% paraformaldehyde fixation followed by permeabilization to facilitate antibody access to the mitochondrial target . Optimization of antibody concentration is also cell-type dependent, with recommendations ranging from 1:50-1:200 for IF/ICC applications .

What are the implications of TXN2 haplodeficiency on cellular antioxidant defenses?

Studies investigating TXN2 haplodeficiency provide valuable insights into the role of this protein in cellular antioxidant defense mechanisms. While complete knockout of TXN2 in mice results in embryonic lethality at E10.5-12.5 with neural tube defects and massive apoptosis, heterozygous TXN2 knockout mice (Txn2+/-) are viable . Research examining the effects of Txn2 haplodeficiency on cochlear antioxidant defenses, auditory function, and cochlear cell loss demonstrated that partial reduction in TXN2 levels has tissue-specific effects on redox homeostasis . These models are valuable for investigating the dose-dependent effects of TXN2 in oxidative stress responses.

When designing experiments using Txn2 haplodeficient models, researchers should consider:

• Age-dependent effects, as redox imbalance may accumulate over time

• Tissue-specific consequences of partial TXN2 deficiency

• Potential compensatory upregulation of other antioxidant systems

• Challenge studies with oxidative stressors to reveal phenotypes that may not be apparent under basal conditions

These models provide opportunities to investigate the threshold levels of TXN2 required for normal cellular function in different tissues and under various stress conditions.

How can TXN2 antibodies be used to investigate mitochondrial dysfunction in oxidative stress models?

TXN2 antibodies are valuable tools for investigating mitochondrial dysfunction in oxidative stress models due to TXN2's critical role in mitochondrial redox homeostasis. Multiple experimental approaches can employ TXN2 antibodies for this purpose:

First, immunofluorescence microscopy using TXN2 antibodies can visualize changes in mitochondrial morphology and TXN2 distribution under oxidative stress conditions. The validated protocol using 1:100 antibody dilution in cell lines like U2OS provides clear mitochondrial staining that can reveal stress-induced changes in mitochondrial network architecture . Second, flow cytometry with TXN2 antibodies enables quantitative assessment of TXN2 protein levels across cell populations exposed to different oxidative stressors. This approach has been validated using THP-1 cells with appropriate fixation and permeabilization protocols . Third, Western blot analysis comparing TXN2 expression between control and stressed samples can identify potential adaptive responses in the mitochondrial redox system, with the expected band at approximately 14 kDa .

For comprehensive mitochondrial dysfunction studies, TXN2 antibody-based analyses should be complemented with functional assays such as mitochondrial membrane potential measurements, as TXN2 has been implicated in regulating this parameter . This multi-parameter approach provides mechanistic insights into the role of TXN2 in maintaining mitochondrial integrity under oxidative stress conditions.

What technical challenges exist in detecting post-translational modifications of TXN2?

Detecting post-translational modifications (PTMs) of TXN2 presents several technical challenges that researchers must address for successful analysis. Since TXN2 functions in a dynamic redox environment, oxidative modifications such as disulfide bond formation and glutathionylation are physiologically relevant but methodologically challenging to preserve during sample preparation. To maintain the native redox state, samples should be prepared with alkylating agents (e.g., N-ethylmaleimide or iodoacetamide) to prevent artificial oxidation during lysis.

Western blot detection of modified TXN2 often requires specialized approaches such as:

• Non-reducing gels to preserve disulfide bonds

• Phospho-specific antibodies when studying phosphorylation events

• Special staining techniques for detecting glutathionylation or nitrosylation

For immunoprecipitation of modified TXN2, standard protocols may need optimization to maintain PTMs during the procedure. The small size of TXN2 (13-14 kDa) can make separation of modified forms challenging on standard SDS-PAGE gels, often requiring higher percentage gels (15-20%) for adequate resolution . Additionally, low abundance of specific modified forms may necessitate enrichment strategies prior to detection.

Despite these challenges, studying TXN2 PTMs is valuable for understanding its regulation and function in mitochondrial redox signaling networks.

How can researchers differentiate between TXN1 and TXN2 in experimental settings?

Differentiating between the closely related thioredoxin family members TXN1 (cytosolic) and TXN2 (mitochondrial) requires careful experimental design and specific methodological approaches. The following strategies are effective for distinguishing these proteins:

First, subcellular fractionation followed by Western blot analysis can separate cytosolic (TXN1) and mitochondrial (TXN2) compartments. When performing Western blot, subtle molecular weight differences may be observed, with TXN2 typically detected at approximately 13-14 kDa . Second, immunofluorescence microscopy using specific antibodies for each protein reveals distinct localization patterns: TXN2 co-localizes with mitochondrial markers, while TXN1 shows diffuse cytoplasmic staining. This approach has been validated using TXN2 antibodies in cell lines such as NIH/3T3 and U2OS .

For functional studies, selective knockdown or knockout models targeting either TXN1 or TXN2 can isolate their individual contributions to cellular processes. When selecting antibodies, verify that they were raised against unique epitopes to ensure specificity. The TXN2 antibodies discussed in the search results utilize immunogens corresponding to amino acids 1-166 of human TXN2 (NP_036605.2) , which contains sequences distinct from TXN1. In experimental design, incorporate positive controls with known expression of each protein, and consider including samples with manipulated expression (overexpression or knockdown) of each protein to further validate specificity.

What are the best practices for quantifying TXN2 expression levels across different experimental conditions?

Accurate quantification of TXN2 expression across experimental conditions requires rigorous methodological approaches. For Western blot quantification, the following best practices should be implemented: First, ensure equal protein loading (20-30 μg per lane) verified by both total protein staining and consistent housekeeping protein detection . Second, use appropriate positive controls such as human HEL, THP-1, K562, or 293T cell lysates that consistently express TXN2 . Third, perform technical replicates (minimum of three) and biological replicates to account for technical and biological variability. Fourth, use calibrated image acquisition within the linear dynamic range of detection, avoiding saturated signals.

For flow cytometry quantification of TXN2, standardized protocols involving appropriate fixation with 4% paraformaldehyde and permeabilization to access the mitochondrial protein are essential . Include appropriate isotype controls to establish background staining levels. When performing qPCR for TXN2 mRNA quantification, validate primers for specificity and efficiency, and use multiple reference genes for normalization.

Statistical analysis should incorporate appropriate tests based on data distribution, with non-parametric tests often more suitable for protein expression data. When comparing multiple conditions, adjust for multiple comparisons to maintain statistical rigor. By following these quantification practices, researchers can generate reliable data on TXN2 expression changes in response to experimental manipulations.

What experimental models are most appropriate for studying TXN2's role in disease pathogenesis?

Selecting appropriate experimental models for studying TXN2's role in disease pathogenesis depends on the specific disease context and research questions. Based on TXN2's functions and disease associations, several model systems have proven valuable:

For cancer research, human cancer cell lines with differential TXN2 expression provide accessible models. The validated detection of TXN2 in cell lines such as 293T, THP-1, K562, and HEL makes these useful starting points . Manipulating TXN2 expression through overexpression or knockdown/knockout approaches can reveal its contributions to cancer cell survival, proliferation, and response to oxidative stress or chemotherapeutics.

For neurodegenerative disorders, where mitochondrial dysfunction and oxidative stress are prominent features, both cellular and animal models are relevant. TXN2 haplodeficient mouse models (Txn2+/-) offer insights into the consequences of partial TXN2 deficiency without the embryonic lethality of complete knockout . For cardiovascular diseases, rat and mouse heart tissue models have demonstrated successful TXN2 detection and can be used to study its role in cardiac pathophysiology .

Disease-specific considerations include selecting models that recapitulate key pathological features, using appropriate stressors to challenge the redox system, and incorporating age-dependent factors when relevant. Multi-level approaches combining in vitro, ex vivo, and in vivo models provide complementary insights into TXN2's complex roles in disease mechanisms.

What considerations are important when developing co-immunoprecipitation protocols for TXN2 and its interaction partners?

Developing effective co-immunoprecipitation (co-IP) protocols for TXN2 and its interaction partners requires addressing several key considerations specific to this mitochondrial redox protein. First, lysis buffer composition is critical: Use mild, non-denaturing buffers (e.g., NP-40 or Triton X-100 based) that preserve protein-protein interactions while effectively extracting mitochondrial proteins. Include protease inhibitors and consider phosphatase inhibitors if phosphorylation-dependent interactions are of interest.

Second, since TXN2 functions in redox reactions, preserving the native redox state during lysis is essential. Consider including reducing agents (DTT or β-mercaptoethanol) to maintain reduced cysteines, or alternatively, alkylating agents (NEM or iodoacetamide) to capture the in vivo redox state. The choice depends on whether you're studying redox-dependent or redox-independent interactions.

Third, antibody selection should prioritize antibodies validated for immunoprecipitation applications. Based on the search results, researchers may need to perform additional validation of commercial TXN2 antibodies specifically for co-IP applications, as this application was not explicitly mentioned in the provided validation data .

Fourth, controls must include: (1) Isotype control antibodies to identify non-specific binding; (2) Input samples to verify target protein presence; (3) Reciprocal co-IPs when possible to confirm interactions; and (4) Negative controls lacking predicted interaction partners.

Fifth, detection methods should consider the small size of TXN2 (13-14 kDa) when selecting gel systems, with higher percentage gels (15-20%) providing better resolution in this molecular weight range. Western blot analysis of co-IP samples should use optimized antibody concentrations previously validated for WB applications (1:500-1:2000) .

What are common causes of non-specific binding when using TXN2 antibodies, and how can they be mitigated?

Non-specific binding is a common challenge when working with antibodies, including those targeting TXN2. Several specific causes and mitigation strategies should be considered:

First, inadequate blocking is a frequent issue. To address this, optimize blocking conditions using 5-10% non-fat milk or BSA in TBS/TBST for Western blot applications . For immunohistochemistry and immunofluorescence, blocking with 10% goat serum (or serum from the species of the secondary antibody) has shown good results in validated protocols . Second, excessive primary antibody concentration can increase background. Follow the recommended dilutions: 1:500-1:2000 for Western blot and 1:50-1:200 for IF/ICC applications , with optimization for your specific experimental system.

Third, cross-reactivity with related proteins (particularly TXN1) can occur. To minimize this, select antibodies raised against unique regions of TXN2, such as those using the full-length recombinant protein (amino acids 1-166) as immunogen . Fourth, inappropriate secondary antibody selection or concentration can contribute to background. Ensure the secondary antibody is specific to the host species of the primary antibody (rabbit for the antibodies described in the search results) and use at recommended dilutions (typically 1:5000-1:10000 for Western blot and 1:100 for IF ).

Additional strategies include thorough washing steps between antibody incubations (3-5 washes of 5 minutes each) , pre-adsorption of the antibody with excess target antigen to confirm specificity, and optimizing incubation conditions (overnight at 4°C for primary antibodies in Western blot applications often reduces non-specific binding).

How can researchers address weak or absent TXN2 signal in Western blot applications?

When encountering weak or absent TXN2 signal in Western blot applications, systematic troubleshooting can identify and resolve the underlying issues. First, consider protein extraction efficiency, especially for mitochondrial proteins like TXN2. Ensure your lysis buffer effectively solubilizes mitochondrial membranes; RIPA buffer supplemented with protease inhibitors has proven effective in validated protocols . Sonication or mechanical disruption may improve extraction of mitochondrial proteins.

Second, verify protein transfer efficiency, as small proteins like TXN2 (13-14 kDa) may transfer through the membrane. Use a lower percentage (10-15%) acrylamide gel, reduce transfer time or voltage, and consider using PVDF membrane instead of nitrocellulose for better retention of small proteins. Third, optimize primary antibody conditions: Increase antibody concentration within the recommended range (1:500-1:2000) , extend incubation time (overnight at 4°C), or try different antibody clones if available.

Fourth, enhance detection sensitivity by using more sensitive ECL substrates, increasing exposure time, or switching to fluorescent-based detection methods. Fifth, check for potential protein degradation by adding fresh protease inhibitors during sample preparation and keeping samples cold throughout processing.

If the issue persists, verify TXN2 expression in your sample using positive controls known to express TXN2, such as human HEL, THP-1, K562, or 293T cell lysates . For loading control, load higher protein amounts (30-50 μg) while ensuring equal loading across all samples. Consider enriching for mitochondrial fractions if TXN2 expression is particularly low in your experimental system.

What strategies can improve TXN2 antibody performance in challenging tissue types?

Improving TXN2 antibody performance in challenging tissue types requires tissue-specific optimization strategies. First, adapt antigen retrieval methods based on tissue characteristics. For formalin-fixed paraffin-embedded (FFPE) tissues, heat-mediated antigen retrieval in citrate buffer (pH 6.0) for 20 minutes has proven effective for TXN2 detection in human mammary cancer tissues and mouse small intestine tissues . For tissues with high lipid content, consider adding detergents to enhance penetration.

Fourth, reduce autofluorescence in IF applications: Tissues with high natural fluorescence (liver, kidney, nervous tissue) benefit from additional blocking steps with Sudan Black B or commercial autofluorescence quenching reagents before antibody incubation. Fifth, adjust antibody incubation conditions: For dense tissues, extend primary antibody incubation time (24-48 hours at 4°C) and use lower antibody concentrations with longer incubation rather than higher concentrations for shorter periods.

Additional strategies include using amplification systems (HRP-conjugated polymers or tyramide signal amplification) for tissues with low TXN2 expression, optimization of section thickness (thinner sections improve antibody penetration), and tissue-specific blocking (with normal serum from the species of the secondary antibody) .