TXNDC9 Antibody

What is TXNDC9 and why is it significant in cancer research?

TXNDC9 (thioredoxin domain-containing protein 9) is a protein belonging to the highly-conserved TRX family, which is implicated in multiple biological processes via modulation of oxidative stress response. It has gained significance in cancer research because it is frequently upregulated in multiple cancer types including lung adenocarcinoma (LUAD), colorectal cancer, breast cancer, and hepatocellular carcinoma (HCC). In LUAD specifically, high TXNDC9 expression correlates with poor patient prognosis, suggesting its potential as both a biomarker and therapeutic target. TXNDC9 functions as a tumor promoter by enhancing cell proliferation, migration, and invasion while inhibiting apoptosis in cancer cells . Additionally, TXNDC9 has been shown to have a negative effect on protein folding by reducing the activity of the chaperone protein TCP1 complex ATPase, which impacts actin and tubulin processing .

What are the standard applications for TXNDC9 antibodies in research?

TXNDC9 antibodies are utilized in multiple experimental applications that help researchers investigate this protein's expression and function. The primary applications include:

These applications have been validated in human samples including cancer cell lines such as Raji, HEK-293, K-562, and HeLa cells . When designing experiments using TXNDC9 antibodies, researchers should optimize antibody dilutions for their specific experimental system, as sensitivity may vary between sample types and preparation methods.

How should researchers validate TXNDC9 antibody specificity?

Validating antibody specificity is crucial for generating reliable experimental results. For TXNDC9 antibodies, researchers should implement the following validation strategies:

• Positive and negative controls: Use cell lines known to express TXNDC9 (such as A549 or HeLa cells) as positive controls, and compare against cell lines with TXNDC9 knockdown via siRNA .

• Molecular weight verification: Confirm that the detected band in Western blot corresponds to the expected molecular weight of TXNDC9 (25-27 kDa) .

• Knockdown/knockout validation: Compare antibody signal between wild-type samples and those where TXNDC9 has been silenced using RNA interference techniques. In published research, si-TXNDC9 has been successfully used to validate antibody specificity in A549 lung cancer cells .

• Cross-reactivity testing: If working with non-human samples, verify whether the antibody cross-reacts with the TXNDC9 ortholog in your species of interest, as many commercial antibodies are only validated against human TXNDC9 .

• Multiple detection methods: Confirm TXNDC9 presence using multiple techniques (e.g., Western blot and immunofluorescence) to strengthen confidence in antibody specificity.

What are the optimal storage and handling conditions for TXNDC9 antibodies?

To maintain TXNDC9 antibody functionality and prevent degradation, researchers should adhere to the following storage and handling guidelines:

How can researchers optimize TXNDC9 antibody-based detection in cancer tissues with variable expression levels?

Cancer tissues often exhibit heterogeneous TXNDC9 expression, presenting challenges for reliable detection. Advanced optimization strategies include:

• Signal amplification systems: For tissues with low TXNDC9 expression, employ tyramide signal amplification (TSA) or other amplification systems to enhance detection sensitivity without increasing background.

• Antigen retrieval optimization: Since TXNDC9 may be masked by formalin fixation, systematically test multiple antigen retrieval methods (heat-induced epitope retrieval with citrate buffer at pH 6.0 versus EDTA buffer at pH 9.0) to determine optimal conditions for your specific tissue samples.

• Multiplex immunostaining: Combine TXNDC9 detection with markers of cancer progression (such as MMPs or apoptotic markers like Bcl-2/Bax) that have been shown to correlate with TXNDC9 expression in lung adenocarcinoma . This approach provides contextual information about TXNDC9's role in specific tumor regions.

• Digital pathology quantification: Implement digital image analysis algorithms to quantify TXNDC9 immunostaining objectively across tissue samples, enabling more precise correlation with clinical parameters and molecular features.

• Sequential staining protocols: For tissues where TXNDC9 co-localization with interaction partners (such as YWHAG/14-3-3γ) is of interest, develop sequential staining protocols that allow visualization of multiple proteins without cross-reactivity between detection systems.

• Cell-type specific analysis: Incorporate markers to distinguish cancer cells from stromal components, enabling selective analysis of TXNDC9 expression in neoplastic cells versus the tumor microenvironment.

What are the methodological considerations when using TXNDC9 antibodies to study protein-protein interactions?

TXNDC9 has been shown to interact with proteins like YWHAG (14-3-3γ), which is critical to its function in cancer progression. When investigating such interactions:

• Co-immunoprecipitation (co-IP) optimization: When performing co-IP to verify TXNDC9 binding partners, consider:

  • Antibody orientation: Both forward (anti-TXNDC9 for precipitation) and reverse (anti-YWHAG for precipitation) approaches should be tested to confirm interaction, as was demonstrated in A549 cells .

  • Lysis conditions: Use gentle lysis buffers (such as RIPA buffer) that preserve protein-protein interactions while ensuring efficient extraction.

  • Binding conditions: Incubate with the appropriate antibody (TXNDC9 or potential binding partner) overnight at 4°C followed by 2-hour incubation with Protein G/A agarose beads .

• Proximity ligation assay (PLA): This technique can visualize TXNDC9 interactions with potential binding partners in situ with higher sensitivity than conventional co-localization studies.

• FRET/BRET approaches: For dynamic studies of TXNDC9 interactions, consider fluorescence or bioluminescence resonance energy transfer techniques using tagged versions of TXNDC9 and putative binding partners.

• Mass spectrometry validation: Confirm antibody-based interaction findings with unbiased proteomic approaches to identify the full spectrum of TXNDC9 binding partners.

• Interaction domain mapping: Use truncated versions of TXNDC9 in conjunction with specific antibodies to determine which domains are critical for protein-protein interactions.

How can researchers effectively use TXNDC9 antibodies to investigate its role in cancer progression mechanisms?

TXNDC9 has demonstrated oncogenic properties in multiple cancer types, particularly through its effects on proliferation, migration, and apoptosis. Advanced methodological approaches include:

• Spatial-temporal expression analysis: Use TXNDC9 antibodies in time-course experiments to track expression changes during cancer progression, correlating with:

  • Proliferation markers (Ki-67, PCNA)

  • Invasion/migration markers (MMP2, MMP9)

  • Apoptotic markers (Bcl-2, Bax)

• Functional pathway investigation: Combine TXNDC9 antibody-based detection with analysis of:

  • Cell cycle regulators after TXNDC9 knockdown or overexpression

  • Downstream effectors such as YWHAG/14-3-3γ

  • Apoptotic pathway components

• Subcellular fractionation analysis: Use TXNDC9 antibodies on nuclear, cytoplasmic, and membrane fractions to determine compartment-specific functions and translocation events during cancer progression.

• In vivo model validation: Apply TXNDC9 antibodies in xenograft or genetic mouse models to correlate in vitro findings with in vivo cancer progression, particularly focusing on:

  • Tumor growth dynamics

  • Metastatic potential

  • Response to therapeutic interventions

• Therapeutic targeting assessment: Use TXNDC9 antibodies to monitor changes in expression and localization following treatment with potential therapeutic agents targeting pathways associated with TXNDC9 function.

What are the best approaches for investigating TXNDC9 post-translational modifications using antibodies?

Post-translational modifications (PTMs) often regulate protein function. For studying TXNDC9 PTMs:

• Modification-specific antibodies: When available, use antibodies specific to phosphorylated, acetylated, or otherwise modified TXNDC9.

• 2D gel electrophoresis with antibody detection: This approach can separate TXNDC9 isoforms with different PTMs before Western blot detection.

• IP-MS workflow optimization:

  • Immunoprecipitate TXNDC9 using validated antibodies

  • Process samples for mass spectrometry analysis

  • Identify PTMs through specialized MS workflows

  • Validate findings using different antibodies or functional assays

• PTM enzyme inhibition studies: Use inhibitors of kinases, phosphatases, acetyltransferases, or other PTM enzymes in combination with TXNDC9 antibody detection to assess their impact on TXNDC9 function in cancer cells.

• Site-directed mutagenesis validation: Generate TXNDC9 constructs with mutations at putative PTM sites and use antibodies to compare their function with wild-type TXNDC9.

How can researchers address contradictory findings in TXNDC9 antibody-based studies across different cancer types?

Researchers investigating TXNDC9 across different cancers may encounter seemingly contradictory results. Methodological approaches to resolve these include:

• Comprehensive antibody validation: Verify that the same epitope is being detected across studies by:

  • Comparing antibody performance across multiple cancer cell lines

  • Using multiple antibodies targeting different TXNDC9 epitopes

  • Confirming specificity in each experimental system

• Context-dependent analysis: Systematically investigate factors that might explain discrepancies:

  • Cell lineage-specific effects (epithelial vs. mesenchymal)

  • Genetic background variations (mutation status of key oncogenes)

  • Microenvironment influences on TXNDC9 function

• Isoform-specific detection: Determine if cancer-specific TXNDC9 isoforms exist that might be differentially detected by antibodies, potentially explaining functional differences.

• Interaction partner profiling: Use co-IP followed by mass spectrometry to identify if TXNDC9 interacts with different partners in different cancer types, potentially explaining functional variations.

• Meta-analysis methodology: When comparing published studies:

  • Standardize quantification methods across studies

  • Account for differences in antibody clones, dilutions, and detection systems

  • Consider statistical power and sample size variations

• Unified experimental design: When possible, conduct parallel experiments across multiple cancer models using identical antibody-based protocols to directly compare TXNDC9 behavior.