TXN2 Antibody

What is TXN2 and what is its biological function?

TXN2 (Thioredoxin-2) is a mitochondrial member of the thioredoxin family encoded by a nuclear gene. It's also known as TRX2, MTRX, or Mt-TRX. TXN2 plays a crucial role in regulating the mitochondrial redox state and is important for cell proliferation . The protein functions as part of cellular defense mechanisms against oxidative stress within the mitochondria. This nuclear gene encodes a mitochondrial protein that is part of a group of small proteins involved in redox reactions through reversible oxidation of their active center dithiol to a disulfide . TXN2's function is particularly critical for maintaining proper redox homeostasis within mitochondria, which directly impacts cellular energy production and prevents oxidative damage.

What types of TXN2 antibodies are available for research?

Research-grade TXN2 antibodies are available in both polyclonal and monoclonal formats:

• Polyclonal antibodies: These include rabbit polyclonal antibodies that recognize various epitopes across the TXN2 protein. Examples include antibodies generated against recombinant fusion proteins containing amino acids 1-166 of human TXN2 .

• Monoclonal antibodies: These offer higher specificity for a single epitope, such as the monoclonal antibody clone 7B5 mentioned in search result .

The selection between polyclonal and monoclonal depends on the specific research application. Polyclonal antibodies typically provide stronger signals due to recognition of multiple epitopes, while monoclonals offer greater specificity and batch-to-batch consistency for long-term studies.

What is the molecular weight of TXN2 protein?

The molecular weight characteristics of TXN2 are:

• Calculated molecular weight: 18 kDa based on its amino acid sequence (166 amino acids)

• Observed molecular weight: Typically 12-14 kDa in experimental Western blot conditions

This discrepancy between calculated and observed molecular weights is significant and consistent across multiple studies. The difference likely results from post-translational modifications, proteolytic processing of the mature protein after mitochondrial import, or the compact nature of the folded protein affecting its migration pattern in SDS-PAGE. Researchers should expect to observe TXN2 at approximately 12-14 kDa rather than the theoretical 18 kDa when performing Western blot analysis.

Which species reactivity has been confirmed for TXN2 antibodies?

TXN2 antibodies have demonstrated reactivity with samples from multiple species:

• Human: Confirmed reactivity in various cell lines (HEL, THP-1, K562, 293T) and tissues (liver, intestine, mammary, lung)

• Mouse: Validated in heart, kidney, and small intestine tissues

• Rat: Tested in heart, kidney, and intestine tissues

The cross-species reactivity reflects the high conservation of TXN2 protein sequence across mammalian species. When selecting a TXN2 antibody for research, verify that it has been specifically validated for your species of interest, as epitope recognition may vary despite sequence homology.

What are the common applications for TXN2 antibodies?

TXN2 antibodies have been validated for multiple research applications:

• Western Blot (WB): For detecting TXN2 protein expression levels in cell and tissue lysates, typically observing a band at 12-14 kDa

• Immunohistochemistry (IHC): For localizing TXN2 in tissue sections, including paraffin-embedded samples of various cancer and normal tissues

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For visualizing TXN2 subcellular localization in cultured cells, typically showing a mitochondrial distribution pattern

• ELISA: For quantitative detection of TXN2 protein in solution

• Flow Cytometry: For analyzing TXN2 expression at the single-cell level, as demonstrated with THP-1 cells

Each application provides different insights into TXN2 expression, localization, or function, enabling researchers to select the appropriate method based on their specific research questions.

What are the recommended dilutions for TXN2 antibodies in different applications?

Optimal dilutions for TXN2 antibodies vary by application technique:

These recommendations provide starting points, but optimal dilutions may vary depending on the specific antibody, sample type, and detection method. It is emphasized that "optimal dilutions/concentrations should be determined by the end user" and dilutions are often "sample-dependent" . A titration experiment is strongly recommended when using a new antibody or testing a new sample type.

What antigen retrieval methods work best for TXN2 immunohistochemistry?

Heat-mediated antigen retrieval methods have been successfully employed for TXN2 immunohistochemistry:

• Citrate buffer (pH 6.0):

  • Used successfully for multiple tissue types including human intestinal cancer, mammary cancer, lung cancer, and normal mouse and rat intestine tissues

  • Protocol: Heat treatment in citrate buffer for 20 minutes

• TE buffer (pH 9.0):

  • Recommended as an alternative retrieval method with potentially improved epitope accessibility

  • May provide stronger staining for some tissue types

• Enzymatic antigen retrieval:

  • For immunocytochemistry applications, IHC enzyme antigen retrieval reagent (15 minutes treatment) has been effective

The choice between these methods depends on the specific tissue type, fixation method, and particular TXN2 antibody being used. Comparative testing may be necessary to determine which method provides optimal signal-to-noise ratio for specific experimental conditions.

How should TXN2 antibodies be stored for optimal stability?

To maintain TXN2 antibody performance over time, follow these storage recommendations:

• Temperature: Store at -20°C for long-term stability

• Formulation: Most TXN2 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting considerations: For some antibodies, creating small aliquots is recommended to avoid repeated freeze/thaw cycles , while for others, aliquoting is specified as unnecessary for -20°C storage

• Stability period: When stored properly, antibodies typically remain stable for one year after shipment

• Additives: Some preparations may contain 0.1% BSA to enhance stability

Following manufacturer-specific storage instructions is essential for maintaining antibody performance. When working with the antibody, keep it cold (on ice) and return to -20°C promptly after use to maximize shelf life and preserve functionality.

What validation strategies confirm TXN2 antibody specificity?

Comprehensive validation of TXN2 antibody specificity requires multiple complementary approaches:

• Genetic validation approaches:

  • Knockout/Knockdown testing: Several TXN2 antibodies have been validated using KD/KO approaches, which represent the gold standard for specificity confirmation

  • This involves comparing antibody signals between wild-type samples and those where TXN2 has been genetically depleted

• Multiple detection methods:

  • Cross-platform validation: Confirm TXN2 detection across different techniques (WB, IHC, IF) to ensure consistent results

  • Multiple antibody comparison: Cross-validate with different antibodies targeting distinct epitopes of TXN2

• Molecular weight verification:

  • Band size: In Western blot, confirm detection of a band at the expected molecular weight (12-14 kDa for TXN2)

  • Band pattern: Look for a clean, single band rather than multiple non-specific bands

• Expression pattern analysis:

  • Tissue distribution: Compare detection in tissues known to express TXN2 versus low-expression tissues

  • Subcellular localization: Verify mitochondrial localization through co-staining with established mitochondrial markers

• Recombinant protein controls:

  • Positive controls: Use purified recombinant TXN2 protein to confirm antibody reactivity

  • Blocking peptides: Perform competition assays with the immunizing peptide to confirm binding specificity

Implementing multiple validation strategies provides higher confidence in antibody specificity and ensures reliable experimental results when studying TXN2.

How does TXN2 expression differ across cancer tissues compared to normal tissues?

The search results provide evidence of TXN2 detection in various cancer tissues, suggesting potential research directions for examining differential expression:

Cancer tissues showing TXN2 expression:

• Human intestinal cancer

• Human mammary cancer

• Human lung cancer

• Human pancreatic cancer

Normal tissues with TXN2 detection:

• Mouse small intestine

• Rat intestine

• Human liver

For rigorous comparative analysis of TXN2 expression in cancer versus normal tissues, researchers should employ:

• Quantitative methods: Digital image analysis of IHC staining intensity, quantitative Western blot with normalization to loading controls, or qPCR for mRNA expression levels.

• Paired sample analysis: Examining matched tumor and adjacent normal tissue from the same patients to control for inter-individual variation.

• Multi-cancer profiling: Comparing TXN2 levels across diverse cancer types using tissue microarrays to identify cancer-specific expression patterns.

• Correlation with clinical parameters: Analyzing TXN2 expression in relation to cancer stage, grade, and patient outcomes to assess potential prognostic value.

• Subcellular distribution analysis: Determining whether TXN2's mitochondrial localization pattern differs between cancer and normal cells, potentially indicating functional alterations.

These approaches would establish whether TXN2 expression is consistently altered in cancer contexts, possibly revealing its role as a potential biomarker or therapeutic target in specific malignancies.

What experimental designs best elucidate TXN2's role in mitochondrial redox regulation?

To comprehensively investigate TXN2's function in mitochondrial redox regulation, implement these experimental approaches:

• Genetic manipulation strategies:

  • Conditional knockout models: Since complete TXN2 deficiency leads to apoptosis even without external stress , inducible systems provide temporal control of TXN2 depletion

  • Active site mutagenesis: Modifying TXN2's catalytic cysteines to create redox-inactive variants

  • Expression level modulation: Creating cellular models with varied TXN2 expression levels

• Redox state assessment techniques:

  • Mitochondrial ROS measurement: Using mitochondria-targeted redox-sensitive fluorescent proteins (e.g., mito-roGFP) or small-molecule probes

  • Thiol redox proteomics: Identifying oxidation states of TXN2 substrates using mass spectrometry-based approaches

  • Glutathione redox state analysis: Measuring GSH/GSSG ratios in mitochondrial fractions

• Functional outcome measurements:

  • Mitochondrial function parameters: Assessing membrane potential, respiratory chain activity, and ATP production

  • Cell death quantification: Measuring apoptosis rates in cells with TXN2 manipulation, with particular attention to activation of caspase 9 and caspase 3, and cytochrome c release

  • Protein aggregation analysis: Examining whether TXN2 deficiency leads to increased protein misfolding or aggregation

• Stress response characterization:

  • Oxidative stress challenges: Exposing cells to various oxidative stressors with and without TXN2

  • Recovery kinetics: Measuring how quickly redox balance is restored after stress in the presence/absence of TXN2

  • Adaptation mechanisms: Identifying compensatory responses when TXN2 function is compromised

These experimental approaches, when combined with immunodetection using validated TXN2 antibodies , provide a comprehensive framework for understanding TXN2's specific contributions to mitochondrial redox homeostasis.

How can TXN2 antibodies be utilized in multiplex immunofluorescence studies?

Multiplex immunofluorescence (mIF) with TXN2 antibodies enables simultaneous visualization of TXN2 alongside other proteins, providing insights into its localization and potential interactions:

• Antibody selection criteria:

  • Choose TXN2 antibodies explicitly validated for immunofluorescence applications

  • Verify minimal background and specific mitochondrial staining pattern in single-staining experiments before multiplexing

  • Consider antibody host species compatibility with other antibodies in your panel

• Multiplex panel design considerations:

  • Mitochondrial co-markers: Include established mitochondrial markers (TOMM20, COX IV, MitoTracker) to confirm TXN2's mitochondrial localization

  • Functional context markers: Add proteins relevant to TXN2's role (other redox proteins, apoptosis markers, proliferation indicators)

  • Choose fluorophores with minimal spectral overlap for clear discrimination between targets

• Optimized staining protocol based on search results:

  • Cell fixation: 4% paraformaldehyde

  • Permeabilization: Suitable permeabilization buffer for mitochondrial access

  • Blocking: 10% goat serum to minimize non-specific binding

  • Primary antibody incubation: Anti-TXN2 at 2μg/mL overnight at 4°C

  • Secondary detection: Fluorophore-conjugated species-specific secondary antibodies (e.g., DyLight®488 Conjugated Goat Anti-Rabbit IgG)

  • Nuclear counterstain: DAPI for nuclear visualization

• Advanced multiplexing strategies:

  • Sequential staining with antibody stripping for same-species antibody combinations

  • Tyramide signal amplification for enhanced sensitivity and sequential same-species antibody use

  • Direct-conjugated primary antibodies to avoid secondary antibody cross-reactivity

• Analysis approaches:

  • Colocalization quantification: Measuring spatial overlap between TXN2 and other proteins

  • Proximity analysis: Assessing physical closeness between TXN2 and potential interacting partners

  • Subcellular distribution: Analyzing TXN2 distribution patterns under different experimental conditions

Multiplex immunofluorescence provides valuable spatial context for understanding TXN2's function within the cellular environment, enabling detection of changes in localization, interactions, and abundance in response to experimental manipulations or disease states.

How can TXN2 knockout/knockdown models be validated using antibody-based methods?

Rigorous validation of TXN2 genetic manipulation models is essential for ensuring experimental reliability. Antibody-based methods provide multiple validation approaches:

• Western blot validation strategy:

  • Primary detection: Use validated TXN2 antibodies to confirm protein depletion in KO/KD samples

  • Control comparison: Quantify band intensity at 12-14 kDa between control and KO/KD samples

  • Loading controls: Include mitochondrial housekeeping proteins (e.g., VDAC) for normalization

  • Multiple antibody confirmation: Validate with antibodies targeting different TXN2 epitopes

• Immunofluorescence/immunocytochemistry validation:

  • Localization pattern: Confirm loss of mitochondrial TXN2 staining in KO/KD cells

  • Co-staining approach: Include mitochondrial markers to verify specific loss of TXN2 signal while preserving mitochondrial structure

  • Quantitative analysis: Measure fluorescence intensity reduction to assess knockdown efficiency

  • Protocol basis: Adapt IF protocols from search result with appropriate controls

• Flow cytometry validation:

  • Single-cell analysis: Quantify TXN2 protein levels across cell populations

  • Population heterogeneity: Identify potential subpopulations with variable knockdown efficiency

  • Methodology reference: Based on flow cytometry protocol for TXN2 demonstrated with THP-1 cells

• Functional validation complementing antibody methods:

  • Phenotypic confirmation: Assess expected consequences of TXN2 deficiency

  • ROS accumulation: Measure increased intracellular ROS levels

  • Apoptosis markers: Confirm activation of caspase 9 and caspase 3

  • Cytochrome c release: Verify mitochondrial disruption as described in search result

• Rescue experiment design:

  • Re-expression strategy: Reintroduce TXN2 in knockout cells

  • Antibody confirmation: Verify TXN2 protein restoration using the same antibodies used to confirm knockout

  • Functional reversal: Demonstrate normalization of phenotypes upon TXN2 re-expression

These validation approaches ensure that observed phenotypes can be confidently attributed to specific TXN2 depletion rather than off-target effects, providing a solid foundation for subsequent functional studies.

What methods are most effective for studying TXN2 protein-protein interactions?

For investigating TXN2's interaction network, several complementary methods can be employed:

• Antibody-based co-immunoprecipitation (Co-IP):

  • Forward approach: Use TXN2 antibodies to pull down TXN2 and associated proteins

  • Reverse approach: Immunoprecipitate suspected interaction partners and probe for TXN2

  • Controls: Include IgG control, input samples, and validation in TXN2 KO samples

  • Western blot detection: Identify co-precipitated proteins using specific antibodies

• Proximity-based methods for mitochondrial context:

  • BioID/TurboID: Fuse TXN2 to a biotin ligase to biotinylate proximal proteins

  • APEX2: Similar approach using an engineered peroxidase

  • Advantage: These methods are particularly valuable for mitochondrial proteins like TXN2 where interactions may be transient or compartmentalized

• Crosslinking strategies for transient interactions:

  • Chemical crosslinking: Stabilize interactions before immunoprecipitation with TXN2 antibodies

  • Photo-crosslinking: Incorporate photo-activatable amino acids into TXN2

  • Benefit: Captures transient or weak interactions that might be missed by standard Co-IP

• Fluorescence-based interaction visualization:

  • FRET (Förster Resonance Energy Transfer): Measure direct protein proximity in living cells

  • BiFC (Bimolecular Fluorescence Complementation): Visualize interactions through complementary fluorescent protein fragments

  • Application basis: Can build upon immunofluorescence protocols established for TXN2

• Mass spectrometry for unbiased interaction discovery:

  • Immunoprecipitation-mass spectrometry: Pull down TXN2 using validated antibodies followed by MS identification

  • SILAC approaches: Use stable isotope labeling to distinguish specific from non-specific interactions

  • Redox proteomics: Identify proteins that undergo TXN2-dependent redox changes

When studying TXN2 interactions, it's crucial to consider its mitochondrial localization and redox-sensitive nature. Experimental conditions should preserve the native redox environment to capture physiologically relevant interactions, particularly those dependent on the redox state of TXN2's active site cysteines.

What are the challenges in distinguishing TXN2 from other thioredoxin family members?

Differentiating TXN2 from other thioredoxin family proteins presents several technical challenges that require specific experimental strategies:

• Sequence homology complications:

  • Shared motifs: Thioredoxin family members contain conserved active site motifs (typically CXXC)

  • Similar size: Many thioredoxin proteins have comparable molecular weights

  • Resolution approach: Target antibodies to unique regions outside the conserved catalytic domains

• Antibody specificity assurance:

  • Cross-reactivity testing: Validate TXN2 antibodies against recombinant proteins of other thioredoxin family members

  • Epitope selection: Prefer antibodies generated against unique regions of TXN2

  • Validation methods: Use TXN2 knockout samples to confirm absence of signal

• Subcellular localization as a distinguishing feature:

  • Compartmentalization: TXN2 is primarily mitochondrial, while TXN/TRX1 is mainly cytosolic

  • Fractionation approach: Use subcellular fractionation before Western blot analysis

  • Co-localization strategy: In immunofluorescence, co-stain with mitochondrial markers to confirm TXN2's distinct localization pattern

• Molecular weight differentiation strategies:

  • High-resolution gels: Use gradient gels or Tricine-SDS-PAGE for better separation of similarly sized proteins

  • Expected size: Look for TXN2 at its observed molecular weight of 12-14 kDa

  • Multiple antibody approach: Use several antibodies targeting different epitopes to confirm identity

• Genetic approaches for definitive identification:

  • Selective knockdown: Use siRNA/shRNA specific to TXN2 to confirm antibody specificity

  • Gene editing: Employ CRISPR-Cas9 knockout of TXN2 as a definitive negative control

  • KO validation: Several TXN2 antibodies have been validated using knockout approaches

By combining these approaches, researchers can achieve reliable discrimination between TXN2 and other thioredoxin family members, ensuring accurate interpretation of experimental results.

What are the key considerations when selecting a TXN2 antibody for specific research applications?

When selecting a TXN2 antibody for research, consider these critical factors to ensure experimental success:

• Application compatibility:

  • Verify validation data for your specific application (WB, IHC, IF, Flow Cytometry, etc.)

  • Review images showing expected staining patterns for your application

  • Check recommended dilutions for your application, noting they vary significantly (from 1:20 for IHC to 1:2000 for WB)

• Species reactivity requirements:

  • Confirm experimental validation in your species of interest (human, mouse, rat)

  • For novel species applications, consider sequence homology in the epitope region

  • Note that some antibodies show broader cross-reactivity than others

• Clonality considerations:

  • Polyclonal antibodies: Offer broader epitope recognition, potentially higher sensitivity, but batch variation

  • Monoclonal antibodies: Provide consistent epitope targeting with high specificity, ideal for long-term studies

  • Choose based on your need for sensitivity versus specificity

• Validation rigor assessment:

  • Knockout/knockdown validation: The gold standard for specificity

  • Multi-technique validation: Confirmation across multiple applications

  • Publications: Previous successful use in peer-reviewed research

• Technical specifications alignment:

  • Storage buffer compatibility with your experiments

  • Long-term stability requirements

  • Special needs (e.g., BSA-free preparations for certain applications)