TMPRSS3 Antibody

What is TMPRSS3 and why is it important in biomedical research?

TMPRSS3 (Transmembrane Protease, Serine 3) is a type II transmembrane serine protease that belongs to the chymotrypsin family of proteases. It has significant importance in biomedical research due to its roles in:

• Hearing: TMPRSS3 functions as a permissive factor for cochlear hair cell survival and activation at the onset of hearing .

• Cancer biology: It shows overexpression in pancreatic cancer and various other cancer tissues, with expression correlating with metastatic potential .

• Genetic disease: Mutations in the TMPRSS3 gene cause non-syndromic autosomal recessive deafness (DFNB8/10) in humans .

The protein's structural features include a transmembrane domain near the N-terminus, a low-density lipoprotein receptor A domain (binds calcium and LDL), a scavenger receptor cysteine-rich domain (protein-protein interaction), and a C-terminal serine protease domain from the S1 family .

What are the key applications for TMPRSS3 antibodies in research?

TMPRSS3 antibodies are valuable tools for multiple research applications including:

• Western Blotting (WB): For protein expression quantification and molecular weight determination, typically showing bands around 40-49 kDa .

• Immunohistochemistry (IHC): For localization in tissue sections, particularly useful in cancer and hearing research .

• Immunofluorescence (IF/ICC): For subcellular localization studies .

• ELISA: For quantitative detection of TMPRSS3 in solution .

These applications allow researchers to investigate TMPRSS3 expression patterns, functional roles, and associations with disease states in various experimental models.

What types of TMPRSS3 antibodies are available and how do they differ?

Based on the available research resources, several types of TMPRSS3 antibodies exist with distinct characteristics:

The choice between these antibodies depends on the specific experimental requirements, target species, and application needs of the researcher.

What are the optimal protocols for Western blotting with TMPRSS3 antibodies?

For successful Western blotting with TMPRSS3 antibodies, researchers should consider the following protocol recommendations:

• Sample preparation: Mouse cochlear extracts or cell lysates (e.g., HeLa, PC-12, A431) at approximately 10 μg per lane .

• Gel type: 8% SDS-PAGE in Tris/glycine buffer system .

• Protein transfer: Electrophoretic transfer onto nitrocellulose membranes .

• Antibody dilution:

  • For polyclonal antibodies: 1:500-1:1000 dilution range is typically recommended .

  • For monoclonal antibodies: 1:1000 dilution has shown good results .

• Detection: Use appropriate secondary antibodies and chemiluminescence detection systems .

• Expected band size: TMPRSS3 has a calculated molecular weight of approximately 49 kDa, but the observed molecular weight is often around 40 kDa .

Note that optimization may be required for specific sample types and antibodies. Always include appropriate positive controls and molecular weight markers.

How should immunohistochemistry protocols be optimized for TMPRSS3 detection?

For optimal immunohistochemical detection of TMPRSS3 in tissue sections:

• Antigen retrieval: Two methods have shown success:

  • TE buffer at pH 9.0 (preferred method)

  • Citrate buffer at pH 6.0 (alternative method)

• Antibody dilutions:

  • For high sensitivity: 1:20-1:50 range

  • For standard applications: 1:50-1:200 range

  • For high specificity: 1:100-1:500 range

• Positive control tissues: Human skin cancer tissue, human cervical cancer tissue, and human thyroid gland papillary carcinoma have shown positive results .

• Visualization system: Compatible with standard HRP/DAB detection systems.

For human inner ear research, researchers have successfully used super-resolution structured illumination microscopy (SR-SIM) to achieve a lateral resolution of approximately 80 nm, allowing for detailed localization of TMPRSS3 in hair cells .

How can TMPRSS3 antibodies be utilized to study its role in hearing loss mechanisms?

TMPRSS3 antibodies have proven valuable for investigating the mechanisms of hearing loss:

• Localization studies:

  • Super-resolution microscopy reveals TMPRSS3 associates with actin in both inner and outer hair cells .

  • TMPRSS3 localizes to cell surface-associated cytoskeletal bodies (surfoskelosomes) in inner and outer pillar cells and Deiters cells .

  • The protein is present in subcuticular organelles in outer hair cells .

• Functional studies:

  • In Tmprss3 Y260X mutant mice, patch-clamp and proteomic analyses show altered outward K+ currents .

  • KCNMA1 channels (needed for outward-rectifying potassium currents) are absent at the neck of inner hair cells in TMPRSS3 mutant mice .

• Methodological approach:

  • Co-localization with cytoskeletal markers like actin helps identify structural roles.

  • Combined approaches using immunofluorescence and electron microscopy provide comprehensive insights.

  • Quantification of fluorescence intensity in various cellular regions helps determine relative expression levels (see Table 3 below) .

What experimental strategies can help resolve discrepancies between calculated and observed molecular weights of TMPRSS3?

The calculated molecular weight of TMPRSS3 is approximately 49 kDa, but the observed molecular weight in Western blots is often around 40 kDa . This discrepancy requires careful experimental consideration:

• Post-translational modifications analysis:

  • Enzymatic deglycosylation assays can determine if glycosylation contributes to mobility shifts.

  • Phosphatase treatment can assess the impact of phosphorylation on protein migration.

  • Protease inhibitor cocktails during sample preparation can prevent degradation.

• Isoform identification:

  • Use antibodies targeting different epitopes (N-terminal, C-terminal, internal regions) to identify potential isoforms .

  • RT-PCR analysis with primers designed to encompass specific regions (e.g., Y260X mutation) can detect variant transcripts .

  • Sequence verification of PCR products can confirm the presence of specific isoforms .

• Controls and validation:

  • Include recombinant TMPRSS3 protein with known molecular weight.

  • Compare results across different cell lines/tissues.

  • Use knockout/knockdown models (e.g., Tmprss3 Y260X mice) as negative controls .

How can researchers effectively use TMPRSS3 antibodies to investigate its role in cancer progression?

TMPRSS3 was originally identified as a gene overexpressed in pancreatic cancer, with expression correlating with metastatic potential . Researchers can leverage TMPRSS3 antibodies to study its role in cancer through:

• Expression profiling strategies:

  • Tissue microarray analysis with IHC to evaluate expression across cancer types and stages.

  • Quantitative Western blotting to compare expression levels between normal and cancer tissues.

  • Correlation of expression with clinical outcomes and metastatic potential.

• Functional mechanisms investigation:

  • Co-immunoprecipitation to identify binding partners in cancer cells.

  • Immunofluorescence to track subcellular localization changes during cancer progression.

  • Combined with siRNA knockdown or CRISPR editing to assess causality in invasion/metastasis models.

• Specific cancer applications:

  • In pancreatic cancer: Compare expression in SUIT-2 pancreatic cancer cell line derivatives with different metastatic potential .

  • In thyroid carcinoma: IHC analysis has shown positive results in thyroid gland papillary carcinoma .

  • In skin cancer: Multiple antibodies have demonstrated reactivity in human skin cancer tissues .

What are the common pitfalls in TMPRSS3 antibody experiments and how can they be addressed?

Researchers may encounter several challenges when working with TMPRSS3 antibodies:

• Specificity concerns:

  • Solution: Validate with multiple antibodies targeting different epitopes (N-terminal, C-terminal, internal) .

  • Solution: Include appropriate negative controls (knockout/knockdown models if available).

  • Solution: Pre-adsorption of antibody with immunizing peptide to confirm specificity.

• Sensitivity limitations:

  • Solution: Optimize antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) .

  • Solution: Adjust antibody concentration and incubation conditions.

  • Solution: Employ signal amplification methods for low-abundance detection.

• Cross-reactivity issues:

  • Solution: Check predicted reactivity data before selection (e.g., Cow: 93%, Dog: 100%, Guinea Pig: 100%, etc.) .

  • Solution: Validate in species-specific positive control tissues.

  • Solution: Consider using monoclonal antibodies for higher specificity applications .

How can researchers evaluate the reproducibility and reliability of TMPRSS3 antibody results?

Ensuring reproducible and reliable results with TMPRSS3 antibodies requires rigorous validation:

• Multi-method confirmation:

  • Correlate protein detection across different techniques (WB, IHC, IF).

  • Confirm protein expression with mRNA analysis (RT-PCR, RNA-seq).

  • Use orthogonal approaches (e.g., mass spectrometry) to verify identity.

• Technical validation:

  • Include comprehensive positive and negative controls in each experiment.

  • Perform inter-laboratory validation when possible.

  • Document detailed protocols including lot numbers, dilutions, and incubation conditions.

• Antibody qualification:

  • Check for validation data from suppliers (e.g., Western blot images, IHC gallery).

  • Review citation history and published applications.

  • Consider using antibodies with Research Resource Identifiers (RRIDs) for traceability (e.g., RRID: AB_2878470, AB_2881253) .

How might TMPRSS3 antibodies contribute to understanding novel functions beyond hearing and cancer?

While TMPRSS3 is well-studied in hearing loss and cancer, emerging research suggests broader applications:

• Protein-protein interaction networks:

  • Co-immunoprecipitation combined with mass spectrometry can identify novel binding partners.

  • Proximity labeling approaches (BioID, APEX) with TMPRSS3 antibody validation can map interaction landscapes.

  • These methods may reveal unexpected cellular pathways involving TMPRSS3.

• Developmental biology:

  • Temporal expression analysis during cochlear development could reveal critical windows for TMPRSS3 function.

  • Investigation in other developing organs might identify new roles beyond the established functions.

• Potential therapeutic targeting:

  • Antibodies can help validate TMPRSS3 as a therapeutic target in disease models.

  • Screening for inhibitors of TMPRSS3 proteolytic activity could lead to novel therapeutic approaches.

  • Understanding epitope accessibility in living cells may inform antibody-drug conjugate development.

What advanced imaging techniques can enhance TMPRSS3 localization studies using available antibodies?

Recent advances in microscopy offer new opportunities for detailed TMPRSS3 localization:

• Super-resolution approaches:

  • Structured illumination microscopy (SR-SIM) has been successfully used to visualize TMPRSS3 in human inner ear with approximately 80 nm resolution .

  • STORM/PALM methodologies could potentially achieve even higher resolution (10-20 nm).

  • Expansion microscopy could physically enlarge specimens for improved visualization of subcellular structures.

• Dynamic imaging approaches:

  • Live-cell compatible antibody fragments could enable tracking of TMPRSS3 trafficking.

  • FRAP (Fluorescence Recovery After Photobleaching) with fluorescently labeled antibodies could assess protein mobility.

  • Correlative light-electron microscopy could connect immunofluorescence data with ultrastructural context.

• Multiplexed detection:

  • Cyclic immunofluorescence or spectral unmixing can detect TMPRSS3 alongside multiple other markers.

  • Mass cytometry or imaging mass cytometry with metal-conjugated antibodies enables high-parameter analysis.

  • These approaches could reveal co-expression patterns with interacting proteins, providing functional insights.