TMX3 Antibody

What is TMX3 and what is its primary function in cells?

TMX3, also known as Protein Disulfide-Isomerase TMX3, functions as a probable disulfide isomerase that participates in the folding of proteins containing disulfide bonds. It may act as a dithiol oxidase and plays a crucial role in regulating endoplasmic reticulum-mitochondria contact sites through its ability to regulate redox signals . This single-pass membrane protein is localized to the endoplasmic reticulum membrane and contributes to cellular redox homeostasis through its enzymatic activity . The protein contains thioredoxin domains characteristic of the protein disulfide isomerase family, enabling it to catalyze the formation, reduction, and isomerization of disulfide bonds during protein folding.

What alternative names and identifiers are associated with TMX3?

TMX3 is recognized by several alternative names in scientific literature and databases:

These alternative nomenclatures are important to consider when conducting literature searches or database queries to ensure comprehensive coverage of TMX3-related research .

What is the tissue expression pattern of TMX3?

TMX3 exhibits widespread tissue distribution throughout the human body. It is expressed in brain, testis, lung, skin, kidney, uterus, bone, stomach, liver, prostate, placenta, eye, and muscle tissues . This broad expression pattern suggests TMX3 serves fundamental cellular functions across diverse tissue types and physiological systems. Researchers should consider this distribution pattern when selecting appropriate positive control tissues for TMX3 antibody validation experiments.

What criteria should guide my selection of a TMX3 antibody for specific research applications?

When selecting a TMX3 antibody, researchers should evaluate several critical parameters:

The optimal antibody selection should align with your specific experimental design, target species, and detection requirements.

What approaches should I use to validate TMX3 antibody specificity?

A rigorous validation strategy for TMX3 antibodies should include:

• Western blot analysis: Confirm detection of a single band at the predicted molecular weight of 52 kDa in tissues known to express TMX3 . Optimize antibody concentration using recommended dilution ranges (e.g., 1:500-2000 for STJA0008841 ).

• Positive and negative controls: Include tissues/cells with known TMX3 expression levels. Based on expression data, brain, testis, and liver represent appropriate positive controls, while selectively depleted samples should serve as negative controls .

• Knockdown/knockout validation: Use RNA interference or CRISPR-Cas9 techniques to reduce TMX3 expression and confirm corresponding signal reduction.

• Cross-reactivity assessment: Test the antibody against related PDI family members to ensure specificity for TMX3.

• Multiple antibody comparison: Use antibodies targeting different TMX3 epitopes and compare their staining patterns (e.g., N-terminal vs. C-terminal targeting antibodies) .

How do polyclonal and monoclonal TMX3 antibodies compare in research applications?

All TMX3 antibodies identified in the search results are polyclonal rabbit antibodies . While monoclonal alternatives were not identified in the search results, understanding the comparative advantages is important:

For critical research applications, consider evaluating multiple polyclonal antibodies targeting different epitopes to ensure robust and reproducible results.

How can I optimize Western blot protocols for TMX3 detection?

Optimizing Western blot protocols for TMX3 detection requires careful consideration of several parameters:

• Sample preparation: Since TMX3 is an ER membrane protein, use lysis buffers containing appropriate detergents to efficiently solubilize membrane proteins.

• Protein loading and separation: Load 20-50 μg of total protein per lane and separate on 10-12% SDS-PAGE gels to achieve optimal resolution around the 52 kDa mark where TMX3 is expected .

• Transfer conditions: Use standard wet transfer protocols with methanol-containing buffers appropriate for membrane proteins.

• Antibody dilution optimization:

  • STJA0008841: Use at 1:500-2000 dilution

  • ab121414: Use at 1:250 dilution

  • ABIN2377339: Follow manufacturer's recommended dilution

• Positive controls: Include human liver tissue lysate, RT4 cells, or U251 MG cells as positive controls for TMX3 expression .

• Detection method: Use enhanced chemiluminescence (ECL) for sensitive detection, as successfully employed with ab121414 .

• Post-translational modification awareness: Be mindful that N-glycosylation of TMX3 may affect band migration patterns .

What are the recommended protocols for TMX3 immunohistochemistry?

For optimal TMX3 immunohistochemical staining, consider the following protocol recommendations:

• Tissue preparation: Formalin-fixed, paraffin-embedded (FFPE) tissues are compatible with TMX3 immunodetection as demonstrated with ab121414 in human stomach tissue .

• Antibody selection and dilution:

  • STJA0004673: Use at 1:50-1:100 dilution for IHC applications

  • ab121414: Follow manufacturer's recommended dilution for IHC-P applications

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) to unmask epitopes after formalin fixation.

• Detection system: Use detection systems compatible with rabbit primary antibodies, such as polymer-based systems conjugated to HRP.

• Counterstaining: Apply hematoxylin counterstaining to provide cellular context.

• Controls: Include positive control tissues with known TMX3 expression, such as stomach, liver, or brain tissue, alongside negative controls where primary antibody is omitted .

How can TMX3 antibodies be used to investigate ER-mitochondria contact sites?

TMX3's role as a regulator of ER-mitochondria contact sites through redox signal regulation presents interesting research applications:

• Co-immunofluorescence studies: Combine TMX3 antibodies with markers for mitochondria (e.g., TOMM20) and ER (e.g., calnexin) to visualize co-localization at contact sites.

• Proximity ligation assay (PLA): Employ PLA technology to detect and quantify close proximity between TMX3 and proteins at ER-mitochondria interfaces.

• Subcellular fractionation and immunoblotting: Isolate mitochondria-associated ER membranes (MAMs) and analyze TMX3 enrichment using Western blotting.

• Super-resolution microscopy: Utilize structured illumination or STORM microscopy with TMX3 antibodies to visualize nanoscale organization at contact sites.

• Functional studies: Combine TMX3 immunodetection with functional assays measuring calcium transfer or lipid trafficking between organelles.

• Redox state analysis: Pair TMX3 antibody labeling with redox-sensitive probes to correlate TMX3 localization with redox environments at interorganellar junctions.

Why might I observe multiple bands in TMX3 Western blots and how should I interpret them?

Multiple bands in TMX3 Western blots may arise from several biological and technical factors:

When encountering unexpected band patterns, compare results across multiple TMX3 antibodies targeting different epitopes to distinguish between true TMX3 signals and artifacts.

What strategies can address non-specific binding in TMX3 immunodetection?

To minimize non-specific binding and optimize TMX3 signal-to-noise ratio:

• Antibody titration: Systematically test dilutions to identify optimal concentration that maximizes specific signal while minimizing background. Follow manufacturer recommendations:

  • STJA0008841: 1:500-2000 for WB

  • STJA0004673: 1:50-100 for IHC

  • ab121414: 1:250 for WB, 1:10 for IHC-P

• Blocking optimization: Test different blocking agents (BSA, non-fat milk, normal serum) and increase blocking time to reduce non-specific interactions.

• Washing stringency: Increase number and duration of washes with detergent-containing buffers.

• Pre-absorption: Consider pre-absorbing antibodies with non-relevant tissues or recombinant proteins to remove cross-reactive antibodies.

• Secondary antibody controls: Include controls omitting primary antibody to identify secondary antibody-mediated background.

• Sample preparation improvements: Optimize lysis conditions and centrifugation steps to reduce interfering cellular components.

How should TMX3 expression patterns be interpreted across different tissues and experimental conditions?

When analyzing TMX3 expression across tissues or experimental conditions:

• Baseline expression expectations: TMX3 is widely expressed across tissues including brain, testis, lung, skin, kidney, uterus, bone, stomach, liver, prostate, placenta, eye, and muscle . Variations from this pattern may indicate tissue-specific regulation or pathological changes.

• Subcellular localization interpretation: TMX3 should primarily localize to the ER membrane as a single-pass membrane protein . Alterations in this distribution may indicate ER stress, protein misfolding, or changes in ER-mitochondria contact dynamics.

• Quantitative analysis approaches: For Western blots, normalize TMX3 signals to appropriate loading controls and ER markers for accurate comparison between samples.

• Functional correlation: Interpret TMX3 expression changes in context of ER stress markers (BiP/GRP78, CHOP), redox status indicators, or mitochondrial function parameters.

• Control inclusion: Always include appropriate positive control tissues with known TMX3 expression (e.g., liver tissue) alongside experimental samples .

• Validation with orthogonal methods: Confirm key findings using complementary techniques (qPCR, mass spectrometry) to differentiate antibody artifacts from true biological variation.

How can TMX3 antibodies be applied to study protein folding disorders?

TMX3's role as a disulfide isomerase in protein folding makes it relevant to various protein folding disorders:

• Expression analysis in disease models: Compare TMX3 levels in tissues from neurodegenerative disease models, congenital disorders of glycosylation, or other protein folding-related pathologies using quantitative Western blotting.

• Co-immunoprecipitation studies: Use TMX3 antibodies to capture protein complexes and identify client proteins or chaperone interactions that may be dysregulated in folding disorders.

• Protein aggregation correlation: Investigate spatial relationships between TMX3 and protein aggregates in neurodegenerative diseases using dual-label immunofluorescence.

• ER stress response dynamics: Monitor TMX3 expression and localization changes during unfolded protein response activation using time-course experiments and subcellular fractionation.

• Therapeutic intervention assessment: Evaluate how compounds targeting protein folding affect TMX3 expression, localization, or post-translational modifications.

What methodologies can investigate TMX3's role in redox signal regulation?

To study TMX3's function in redox signal regulation:

• Redoxome analysis: Combine TMX3 immunoprecipitation with mass spectrometry to identify proteins forming mixed disulfides with TMX3 under different redox conditions.

• Thiol-disulfide exchange assays: Use purified recombinant TMX3 (identified using TMX3 antibodies) in enzymatic assays to characterize its oxidoreductase activity against potential substrates.

• Redox state-specific antibodies: Develop or utilize antibodies that distinguish between oxidized and reduced forms of TMX3 to monitor its redox state in different cellular contexts.

• Site-directed mutagenesis combined with immunodetection: Analyze how mutations in TMX3's active site cysteines affect its localization and function at ER-mitochondria contact sites.

• Fluorescence lifetime imaging microscopy (FLIM): Combine TMX3 immunolabeling with genetically-encoded redox sensors to correlate TMX3 activity with compartment-specific redox potentials.

How can researchers compare TMX3 with other protein disulfide isomerase family members?

To differentiate TMX3 from other PDI family members:

• Comparative expression analysis: Use antibodies against TMX3 and other PDI family members (PDI, ERp57, ERp72, etc.) to create tissue expression profiles using multiplex immunohistochemistry or Western blotting.

• Subcellular co-localization studies: Perform dual-label immunofluorescence to compare the precise subcellular distribution of TMX3 versus other PDI family members, with particular attention to membrane association patterns.

• Functional complementation experiments: In TMX3 knockdown systems, test whether overexpression of other PDI family members can rescue phenotypes, and use TMX3 antibodies to confirm knockdown efficiency.

• Substrate specificity analysis: Use immunoprecipitation with TMX3 antibodies followed by mass spectrometry to identify unique TMX3 client proteins versus those of other PDI family members.

• Redox potential measurement: Determine the reduction potential of TMX3's active site compared to other PDI family members using biochemical approaches with purified proteins.