TMED3 Antibody

What are the validated applications for TMED3 antibodies?

TMED3 antibodies have been validated for multiple research applications including:

• Western Blot (WB)

• Immunohistochemistry (IHC)

• Immunocytochemistry/Immunofluorescence (ICC-IF)

• Enzyme-Linked Immunosorbent Assay (ELISA)

The validation data shows consistency across these applications with specific reactivity to human, mouse, and rat samples . When designing experiments, researchers should consider that different applications may require specific antibody preparations and optimization protocols.

What are the recommended dilution ratios for TMED3 antibodies in different applications?

The optimal dilution varies by application technique:

Note: It is strongly recommended that each researcher titrate the antibody in their specific experimental system to obtain optimal results . Dilution requirements may vary based on the specific tissue or cell type being analyzed.

What is the observed molecular weight of TMED3 in Western blot applications?

The calculated molecular weight of TMED3 is 25 kDa (217 amino acids), but the observed molecular weight in Western blot applications typically ranges between 21-25 kDa . This slight variation may reflect post-translational modifications or protein processing differences across tissue types. When analyzing Western blot results, researchers should be aware that slight variations in molecular weight may occur depending on cell or tissue type.

What cell lines or tissues show consistent TMED3 expression for use as positive controls?

Based on published research, these samples show reliable TMED3 expression for positive controls:

For IHC applications specifically, human stomach cancer tissue has shown reliable results. When performing antigen retrieval for these samples, TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may serve as an alternative .

How should TMED3 knockdown experiments be designed to study its function in cancer progression?

When designing TMED3 knockdown experiments:

• Vector Selection: Lentiviral vectors have shown efficacy for TMED3 knockdown with MOI=10 reported as effective .

• Transfection Protocol: Polybrene (6 μg/ml) has been successfully used to enhance lentiviral transduction .

• Validation Method: RT-qPCR and Western blot should be performed 72 hours post-transduction to confirm knockdown efficiency .

• Functional Assays: Design experiments to assess:

  • Cell viability (via CCK-8 or similar assays)

  • Migration capacity (transwell and wound healing assays)

  • Apoptosis (flow cytometry)

  • Colony formation ability

Research has shown that TMED3 knockdown inhibits cell viability and migration while enhancing apoptosis in cancer cells, particularly in chordoma and lung squamous cell carcinoma .

What molecular mechanisms mediate TMED3's role in cancer progression?

TMED3 influences cancer progression through multiple molecular pathways:

• Apoptosis Regulation: TMED3 knockdown inhibits expression of anti-apoptotic proteins including:

  • Bcl-2

  • Heat shock protein 27

  • Insulin-like growth factors I and II (IGF-I, IGF-II)

• Cell Cycle Regulation: TMED3 affects cell cycle-related proteins:

  • Akt

  • CDK6

  • Cyclin D1

• Wnt/β-catenin Signaling: In breast cancer, TMED3 promotes proliferation and migration through Wnt/β-catenin signaling pathway activation .

• EMT Regulation: TMED3 knockdown inhibits epithelial-mesenchymal transition in lung squamous cell carcinoma, reducing migration capacity .

When investigating these pathways, researchers should design experiments to examine specific protein interactions rather than assuming a universal mechanism across all cancer types.

How does TMED3 interact with other TMED family proteins, and what experimental approaches are best to study these interactions?

TMED3 forms functional complexes with other TMED family proteins:

• TMED Complexes: Research has identified that TMED3 forms a heteromeric complex with TMED2, TMED9, and TMED10 that facilitates unconventional protein secretion .

• Experimental Approaches:

  • Co-immunoprecipitation assays have successfully demonstrated that TMED3 primarily recognizes cargo proteins, while other TMEDs may facilitate transport

  • Immunofluorescence analysis showing >70% colocalization of TMED3 with cargo proteins under blocked ER-to-Golgi transport conditions

  • Cell surface biotinylation assays can detect TMED3 trafficking under different conditions

• TMED Silencing: When studying TMED family interactions, systematic silencing of individual family members (TMED2, TMED3, TMED9, TMED10) provides insights into their relative contributions to cellular processes .

What is TMED3's role in ER stress-associated unconventional protein secretion, and how can this be experimentally validated?

TMED3 plays a critical role in ER stress-associated unconventional protein secretion (UPS):

• Cargo Recognition: TMED3 specifically recognizes ER core-glycosylated protein cargos during ER stress .

• Glycosylation Preferences: Experimental evidence indicates TMED3 preferentially binds to:

  • Deglucosylated forms of N-glycans (inhibition by 1-deoxynojirimycin reduces TMED3-cargo interaction)

  • High-mannose glycans (mannosidase inhibitor kifunensine increases TMED3-cargo interaction)

• Experimental Validation Approaches:

  • Pull-down assays with ER glucosidase and mannosidase inhibitors

  • ARF1-Q71L-induced UPS models

  • Colocalization studies with cargo proteins under ER stress conditions

• TMED Complex Formation: The TMED2/3/9/10 heteromeric complex is essential for efficient UPS, with TMED3 serving as the initial cargo recognition component .

What are the optimal storage and handling conditions for TMED3 antibodies?

For maximum stability and performance:

• Storage Temperature: Store at -20°C

• Buffer Composition: PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Stability: Stable for one year after shipment when properly stored

• Aliquoting: For 20μL sizes containing 0.1% BSA, aliquoting is unnecessary for -20°C storage

• Shipping Conditions: Typically shipped on wet ice

Following these storage recommendations ensures antibody stability and consistent experimental results.

How can researchers validate the specificity of TMED3 antibodies in their experimental system?

To ensure antibody specificity:

• Positive Controls: Use validated cell lines with known TMED3 expression (HeLa, MCF-7, HepG2)

• Knockdown/Knockout Validation:

  • Compare antibody staining between wild-type and TMED3 knockdown/knockout samples

  • Multiple publications have validated antibody specificity using this approach

• Peptide Competition Assay: Pre-incubate antibody with immunogen peptide (DPQGNTIYRETKKQYDSFTYRAEVKGVYQF for some antibodies) to block specific binding

• Multiple Antibody Comparison: Use antibodies targeting different epitopes of TMED3 to confirm consistency in results

• Cross-Reactivity Assessment: Test antibody reactivity in samples where TMED3 is not expected to be expressed

What special considerations should be taken when performing IHC with TMED3 antibodies?

For optimal IHC results with TMED3 antibodies:

• Antigen Retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

• Tissue Processing:

  • Human stomach cancer tissue has been validated for positive staining

  • Consider tissue-specific fixation effects on epitope accessibility

• Dilution Optimization:

  • Start with the recommended 1:50-1:500 dilution range

  • Perform a dilution series to determine optimal concentration for specific tissue types

• Background Reduction:

  • Include appropriate blocking steps to minimize non-specific binding

  • Consider endogenous peroxidase quenching if using HRP-conjugated detection systems

How does TMED3 expression vary across cancer types, and what implications does this have for its potential as a biomarker?

TMED3 expression patterns vary significantly across cancer types:

• Upregulation Patterns:

  • Breast cancer: Significantly increased TMED3 mRNA and protein expression compared to normal controls; correlated with poor prognosis

  • Lung squamous cell carcinoma: Upregulated expression positively correlated with pathological grade

  • Colorectal cancer: Expression independently associated with prognosis

  • Chordoma: Highly expressed compared to normal tissue

• Biomarker Potential:

  • Prognostic indicator: High TMED3 expression correlates with poor outcomes in breast cancer and colorectal cancer

  • Pathological correlation: Expression levels correlate with pathological grade in lung squamous cell carcinoma

• Research Implications:

  • Different antibody dilutions may be required for different cancer types

  • Validation of expression patterns should be performed for each cancer type under investigation

  • Consider using tissue microarrays with multiple cancer types for comparative studies

What contradictions exist in the literature regarding TMED3 function across different cancer types?

Notable contradictions have been observed:

• Metastasis Regulation:

  • In colon cancer: TMED3 knockdown promoted lung metastasis in mice, suggesting a suppressive role in metastasis

  • In breast cancer: TMED3 overexpression promoted migration and invasion, indicating a pro-metastatic function

• Experimental Considerations:

  • Different experimental models (cell lines, animal models)

  • Variations in knockdown/overexpression techniques

  • Cancer-type specific microenvironments

• Resolution Approaches:

  • Direct comparison studies using identical methodologies across cancer types

  • Investigation of tissue-specific interacting partners

  • Analysis of TMED3 mutations or isoforms across cancer types

When designing experiments, researchers should account for these contradictions and include appropriate controls specific to their cancer type of interest.

What novel applications of TMED3 antibodies might advance understanding of its role in cellular trafficking?

Emerging research areas include:

• Unconventional Protein Secretion:

  • Tracking TMED3's role in ER stress-associated secretion of transmembrane proteins

  • Investigating TMED3's cargo selectivity mechanisms for high-mannose glycans

• Super-Resolution Microscopy:

  • Using advanced imaging techniques with TMED3 antibodies to visualize trafficking dynamics in real-time

  • Co-localization studies with other vesicular transport components

• TMED Complex Formation:

  • Structural studies of TMED2/3/9/10 heteromeric complexes

  • Investigation of assembly/disassembly dynamics under different cellular conditions

• Therapeutic Applications:

  • Exploring TMED3 as a potential therapeutic target for cancer treatment

  • Development of small molecule inhibitors targeting TMED3-cargo interactions

How might single-cell analysis techniques advance our understanding of TMED3 function in heterogeneous tumor environments?

Single-cell approaches offer new insights:

• Single-Cell RNA-seq:

  • Mapping TMED3 expression heterogeneity within tumors

  • Correlating TMED3 expression with specific cell states or phenotypes

• Mass Cytometry:

  • Simultaneous analysis of TMED3 with multiple cancer markers at single-cell resolution

  • Identification of TMED3 expression in specific tumor subpopulations

• Spatial Transcriptomics:

  • Mapping TMED3 expression within the tumor microenvironment

  • Correlating expression with stromal interactions or invasive fronts

• Methodological Considerations:

  • Optimization of TMED3 antibodies for single-cell applications

  • Development of multiplexed antibody panels including TMED3

  • Fixation and permeabilization protocols that preserve both TMED3 epitopes and cellular architecture