txndc17 Antibody

What is TXNDC17 and why is it important to study?

TXNDC17, also known as TRP14 or TXNL5, belongs to the thioredoxin family and functions as a disulfide reductase. It is a widely expressed 123-amino acid protein with a WCPDC motif, featuring a thioredoxin fold and a TRX-like active-site sequence . TXNDC17 plays crucial roles in redox regulation and protein folding, contributing to cellular homeostasis. Research interest has grown due to its potential involvement in diseases related to redox dysregulation, including cancer, neurodegenerative disorders, and cardiovascular diseases . Studying TXNDC17 using specific antibodies enables researchers to understand its expression patterns, localization, interactions, and functional roles in normal and pathological states.

What applications are TXNDC17 antibodies validated for?

TXNDC17 antibodies have been validated for multiple research applications across different experimental systems. Based on available data, these antibodies can be reliably used in the following applications:

It is important to note that optimal dilutions may vary depending on the specific experimental conditions and sample types, so researchers should perform titration experiments to determine ideal concentrations for their specific systems .

What species reactivity can be expected from commercially available TXNDC17 antibodies?

Available TXNDC17 antibodies demonstrate varying species reactivity profiles that researchers should consider when selecting an appropriate antibody for their experiments:

When working with non-human models, researchers should verify cross-reactivity, as some antibodies may recognize conserved epitopes across species while others are strictly species-specific. The antibody from Proteintech offers the broadest cross-species application potential based on available validation data .

How can I optimize Western blot protocols specifically for TXNDC17 detection?

Optimizing Western blot protocols for TXNDC17 detection requires consideration of several technical factors:

The expected molecular weight of TXNDC17 is approximately 14 kDa (123 amino acids), which has been consistently observed in experimental validation . When running gels, consider using higher percentage gels (15-18%) to achieve better resolution of this low molecular weight protein. For protein extraction, standard lysis buffers containing protease inhibitors are generally sufficient, but adding reducing agents is crucial due to TXNDC17's involvement in redox processes.

For primary antibody incubation, begin with manufacturer-recommended dilutions (e.g., 1:5000 for Proteintech's antibody), but be prepared to optimize further based on signal intensity and background. Extended overnight incubation at 4°C often yields superior results compared to shorter incubations at room temperature. Secondary antibody selection should match the host species of the primary antibody (rabbit for most available TXNDC17 antibodies) .

To ensure specificity, include both positive controls (cell lines with known TXNDC17 expression such as HEK-293, HeLa, or A549) and negative controls (primary antibody omission or pre-immune serum) . For challenging experiments, consider signal enhancement systems or more sensitive detection methods such as chemiluminescence with extended exposure times.

What are the potential causes of unexpected bands or inconsistent results when using TXNDC17 antibodies?

When encountering unexpected bands or inconsistent results with TXNDC17 antibodies, several potential issues should be systematically investigated:

Post-translational modifications may result in molecular weight shifts. While the calculated molecular weight of TXNDC17 is 14 kDa, modifications such as phosphorylation or ubiquitination could cause mobility shifts on SDS-PAGE. Additionally, TXNDC17 may form complexes with interaction partners that resist complete denaturation, resulting in higher molecular weight bands.

Cross-reactivity with other thioredoxin family members should be considered, as TXNDC17 belongs to the thioredoxin family which shares structural similarities. Antibody specificity varies between manufacturers and even between lots. To address this, researchers can validate specificity using knockdown/knockout controls or peptide competition assays.

Sample preparation issues frequently contribute to inconsistencies. Incomplete reduction of disulfide bonds (particularly relevant for TXNDC17 given its redox function) or protein degradation during extraction could cause anomalous bands. Using fresh reducing agents and maintaining a comprehensive protease inhibitor cocktail can minimize these issues.

For inconsistent results between experiments, standardize loading controls, antibody lots, and incubation conditions. Document all experimental variables to identify potential sources of variability and adjust protocols accordingly.

How can I design experiments to study TXNDC17's role in redox regulation pathways?

Investigating TXNDC17's role in redox regulation pathways requires a multifaceted experimental approach:

Expression modulation studies: Use siRNA/shRNA for knockdown or CRISPR-Cas9 for knockout of TXNDC17, followed by assessment of cellular redox state using fluorescent redox sensors, glutathione measurements, or protein thiol assays. Complementary overexpression studies can provide additional insights into gain-of-function effects.

Interaction studies: Identify TXNDC17 interaction partners through co-immunoprecipitation using validated TXNDC17 antibodies (e.g., Proteintech's antibody at 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate) , followed by mass spectrometry or Western blot analysis. Proximity ligation assays can further validate these interactions in situ.

Functional assays: Measure enzymatic activity using purified TXNDC17 and potential substrates to determine substrate specificity and kinetics. For cellular studies, expose cells to oxidative stress inducers (H₂O₂, paraquat, or thiol-reactive compounds) and compare responses between TXNDC17-manipulated and control cells.

Localization studies: Use immunofluorescence with TXNDC17 antibodies at proper dilutions (1:50-1:500) to track subcellular localization under different redox conditions. Co-localization with known redox-sensitive proteins can provide functional context.

Pathological model integration: Incorporate these approaches into disease models relevant to redox dysregulation (e.g., neurodegenerative disease models, cancer cell lines, or cardiovascular stress models) to establish physiological significance.

What controls should be included when using TXNDC17 antibodies for immunohistochemistry or immunofluorescence?

Implementing appropriate controls for immunohistochemistry (IHC) or immunofluorescence (IF) studies is essential for generating reliable and interpretable data when using TXNDC17 antibodies:

Positive tissue/cell controls: Include samples known to express TXNDC17, such as HeLa or HepG2 cells which have been positively validated for IF/ICC applications . For tissue sections, human kidney, uterus, or mouse pancreas tissues have demonstrated reliable TXNDC17 expression .

Negative controls: Perform parallel staining omitting the primary antibody while maintaining all other steps to assess secondary antibody specificity and autofluorescence. Additionally, use tissues or cells with confirmed absence or knockdown of TXNDC17 when possible.

Absorption/competition controls: Pre-incubate the TXNDC17 antibody with excess immunizing peptide (when available from manufacturers) before applying to samples. This should significantly reduce or eliminate specific staining.

Counterstains and co-localization markers: Include nuclear counterstains (DAPI or Hoechst) for structural context and consider co-staining with markers of subcellular compartments where TXNDC17 is expected to localize based on its known functions in redox regulation.

Non-specific binding controls: Include isotype-matched control antibodies (rabbit IgG for most TXNDC17 antibodies) to distinguish specific staining from potential Fc receptor binding or other non-specific interactions.

Autofluorescence quenching: For tissues with high autofluorescence, implement appropriate quenching steps before antibody application and include unstained control sections to assess background signal levels.

How should researchers approach the quantification of TXNDC17 expression levels across different experimental conditions?

Accurate quantification of TXNDC17 expression requires methodological rigor and standardization across multiple analytical platforms:

Western blot quantification: Use densitometry with appropriate normalization to loading controls (β-actin, GAPDH, or total protein staining). For TXNDC17, which is a small protein (14 kDa) , ensure sufficient separation time during electrophoresis and consider specialized low-molecular-weight loading controls. Always work within the linear range of detection by running dilution series of representative samples.

qPCR analysis: Design and validate primers specific to TXNDC17 mRNA, using multiple reference genes for normalization. Confirm primer specificity through melt curve analysis and sequencing of amplicons. Calculate relative expression using the 2^-ΔΔCt method or absolute quantification with standard curves.

Immunofluorescence quantification: For microscopy-based quantification, acquire images with consistent exposure settings across all experimental conditions. Perform automated analysis using software that can define regions of interest and measure intensity parameters while controlling for background. Report data as integrated density or mean fluorescence intensity normalized to cell number or area.

Flow cytometry: For cell suspension-based quantification, optimize fixation and permeabilization protocols for intracellular TXNDC17 staining. Include fluorescence-minus-one controls and isotype controls. Report data as median fluorescence intensity with appropriate statistical analysis.

Statistical considerations: Apply appropriate statistical tests based on data distribution, and include sufficient biological and technical replicates (minimum n=3 for both). Report both effect size and statistical significance to provide complete interpretation of TXNDC17 expression changes.

What are the recommended approaches for studying TXNDC17's interaction with other proteins in redox pathways?

To comprehensively characterize TXNDC17's protein interactions within redox regulatory networks, researchers should consider multiple complementary techniques:

Co-immunoprecipitation (Co-IP): Use validated TXNDC17 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) under native conditions to preserve protein-protein interactions. Consider both forward and reverse Co-IP approaches, where either TXNDC17 or its suspected interaction partner serves as the primary target. Given TXNDC17's involvement in redox processes, maintain reducing conditions during lysis and IP procedures to preserve physiologically relevant interactions.

Proximity ligation assay (PLA): This technique can detect protein interactions in situ with high sensitivity and specificity. Use antibodies against TXNDC17 and potential interaction partners from different host species, followed by species-specific PLA probes. This approach is particularly valuable for detecting transient interactions that might be lost during Co-IP procedures.

Bimolecular fluorescence complementation (BiFC): For live-cell analysis of protein interactions, construct fusion proteins of TXNDC17 and suspected partners with complementary fragments of fluorescent proteins. Interaction brings these fragments together, restoring fluorescence that can be visualized microscopically.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can map interaction interfaces between TXNDC17 and its partners by identifying regions protected from deuterium exchange when complexes form. This provides structural insights into the molecular basis of interactions.

Functional validation: Beyond identifying interactions, validate their functional significance through mutagenesis of key residues in the interaction interface, followed by assessment of downstream redox-dependent processes or enzymatic activities.

How can TXNDC17 antibodies be utilized in studies of oxidative stress-related diseases?

TXNDC17 antibodies can be strategically employed in investigating oxidative stress-related pathologies through several specialized approaches:

Tissue expression profiling: Use immunohistochemistry with validated TXNDC17 antibodies to compare expression patterns between normal and diseased tissues. This has particular relevance for neurodegenerative disorders, cardiovascular diseases, and cancers where redox imbalance is a pathogenic factor. Quantitative analysis of staining intensity and localization changes can provide insights into disease mechanisms.

Biomarker development: Explore TXNDC17 as a potential biomarker by analyzing its expression levels in patient samples using techniques like ELISA (using antibody dilutions of 1:5000-1:10000) or Western blotting. Correlate expression with disease severity, progression, or treatment response to establish clinical relevance.

Therapeutic target assessment: In drug discovery pipelines, use TXNDC17 antibodies to monitor how potential therapeutic compounds affect TXNDC17 expression or activity. This is especially relevant for compounds designed to modulate redox pathways.

Genetic modification models: In disease models with genetic manipulation of TXNDC17, use antibodies to confirm knockdown/knockout efficiency or overexpression levels, correlating these with phenotypic outcomes to establish causality rather than mere association.

Post-translational modification analysis: Investigate how oxidative stress affects TXNDC17 through post-translational modifications by combining immunoprecipitation with TXNDC17 antibodies followed by mass spectrometry or modification-specific detection methods.

Co-morbidity studies: In complex disease presentations with multiple co-morbidities, use TXNDC17 antibodies to investigate how this protein might serve as a common mechanistic link through its redox regulatory functions.

What methodological approaches can resolve contradictory findings when studying TXNDC17 across different experimental systems?

When researchers encounter contradictory results regarding TXNDC17 function or expression across different experimental systems, several methodological strategies can help resolve these discrepancies:

Antibody validation consistency: Ensure all laboratories are using validated TXNDC17 antibodies with confirmed specificity. Cross-laboratory validation using the same antibody lot (with attention to catalog numbers and RRID identifiers such as AB_10695499) can eliminate antibody variability as a source of discrepancy.

Standardized protocols: Develop and share detailed protocols for key techniques including sample preparation, Western blotting, immunoprecipitation, and immunofluorescence. Specify critical parameters such as buffer compositions, incubation times/temperatures, and equipment settings.

Cell line authentication: Verify the identity and genetic stability of cell lines used across studies through short tandem repeat profiling or other authentication methods, as genetic drift can alter protein expression patterns and cellular responses.

Physiological context consideration: Systematically analyze how experimental conditions (2D versus 3D culture, oxygen tension, passage number, confluence level) affect TXNDC17 expression and function. Document these parameters comprehensively in publications.

Integrative multi-omics approach: Combine proteomics, transcriptomics, and functional assays to build a comprehensive understanding of TXNDC17's role. This can reveal context-dependent functions that explain apparent contradictions.

Meta-analysis methodology: For published contradictory findings, conduct formal meta-analyses with clearly defined inclusion criteria and quality assessment, potentially revealing patterns in methodology that explain divergent results.

Collaborative cross-laboratory validation: Organize multi-center studies where identical samples are processed and analyzed in different laboratories using standardized protocols, directly addressing reproducibility challenges.

How might emerging antibody technologies enhance TXNDC17 research beyond conventional applications?

Emerging antibody technologies offer exciting opportunities to advance TXNDC17 research beyond traditional applications, opening new avenues for understanding its functions and roles in disease:

Single-domain antibodies (nanobodies): These smaller antibody fragments can access epitopes that conventional antibodies cannot reach, potentially revealing previously undetectable conformational states of TXNDC17, particularly during its catalytic cycle as a disulfide reductase. Their small size also enables superior tissue penetration for in vivo imaging applications.

Intrabodies and chromobodies: Genetically encoded antibody-based sensors can track TXNDC17 in living cells in real-time. By fusing TXNDC17-specific binding domains with fluorescent proteins, researchers can monitor dynamic changes in localization and interaction partners during redox fluctuations or stress responses.

Bi-specific antibodies: These engineered constructs can simultaneously bind TXNDC17 and another protein of interest, potentially forcing or detecting interactions between TXNDC17 and suspected redox partners. This approach could reveal transient or weak interactions that conventional methods might miss.

Antibody-drug conjugates: While primarily developed for therapeutic applications, this technology can be adapted for research by conjugating TXNDC17 antibodies with small molecules that selectively inhibit or activate the protein upon binding, enabling precise temporal control of TXNDC17 function.

Antibody-guided CRISPR/Cas systems: By conjugating TXNDC17 antibodies to Cas9 or other effector proteins, researchers can achieve targeted genomic or epigenomic modifications at sites where TXNDC17 is bound to DNA or chromatin, revealing potential roles in gene regulation.

Mass cytometry (CyTOF) with metal-conjugated antibodies: This technique enables simultaneous detection of dozens of proteins, allowing researchers to place TXNDC17 within complex signaling networks across heterogeneous cell populations with single-cell resolution.

What are the recommended approaches for studying tissue-specific variations in TXNDC17 expression and function?

Understanding tissue-specific variations in TXNDC17 expression and function requires integrative approaches that account for physiological context:

Tissue microarray analysis: Use validated TXNDC17 antibodies to perform immunohistochemistry on tissue microarrays containing multiple tissue types from multiple donors. This allows for systematic comparison of expression patterns while controlling for technical variables. Quantitative image analysis should assess both expression levels and subcellular localization patterns.

Single-cell transcriptomics integration: Correlate immunohistochemical findings with single-cell RNA-seq data to identify cell type-specific expression patterns within heterogeneous tissues. This can reveal specialized functions in specific cell populations that might be masked in whole-tissue analyses.

Tissue-specific knockout models: Generate conditional knockout models where TXNDC17 is deleted in specific tissues of interest. Use TXNDC17 antibodies to confirm knockout efficiency and analyze phenotypic consequences to determine tissue-specific functional requirements.

Comparative interactome mapping: Perform immunoprecipitation with TXNDC17 antibodies (using established protocols of 0.5-4.0 μg antibody per 1.0-3.0 mg lysate) followed by mass spectrometry to identify tissue-specific interaction partners. This can reveal how TXNDC17 functions may be redirected by the tissue-specific protein milieu.

Ex vivo tissue culture systems: Utilize precision-cut tissue slices or organoids to maintain native tissue architecture while enabling experimental manipulation. This approach bridges the gap between in vitro cell cultures and in vivo animal models, allowing for tissue-specific functional studies in a controlled environment.

Physiological stress responses: Compare how TXNDC17 expression and localization respond to relevant physiological stressors (oxidative stress, hypoxia, nutrient deprivation) across different tissue types, potentially revealing tissue-specific adaptive mechanisms.