TMEM31 Antibody

What is TMEM31 and why is it relevant for immunological research?

TMEM31 is a transmembrane protein involved in intracellular trafficking and membrane dynamics. It has gained significant research interest due to its classification as a cancer/testis (CT) antigen - proteins predominantly expressed in normal testis tissue but aberrantly expressed in various malignancies . TMEM31 shows restricted expression in healthy tissues (primarily testis) but elevated expression in certain cancers, particularly during melanoma metastasis . This expression pattern makes it a promising target for cancer immunotherapy, diagnostic development, and biological investigations into cancer progression mechanisms.

The protein plays a role in several cellular processes and may have implications in various pathological conditions including cancer, neurodegenerative disorders, and metabolic diseases . Recent studies have identified TMEM31 as particularly relevant in melanoma research, with expression levels increasing during metastatic progression .

What are the primary applications of TMEM31 antibodies in research?

TMEM31 antibodies are utilized across multiple experimental techniques:

These applications enable researchers to detect, localize, and quantify TMEM31 expression in diverse experimental settings, from tissue sections to cell cultures and protein extracts .

How should researchers select an appropriate TMEM31 antibody for specific experimental applications?

When selecting a TMEM31 antibody, researchers should consider:

• Antibody type: Most available TMEM31 antibodies are polyclonal, derived from rabbits immunized with recombinant human TMEM31 protein (typically amino acids 1-118) . Polyclonals offer high sensitivity but potentially lower specificity than monoclonals.

• Validated applications: Confirm the antibody has been validated for your intended application. Review published validation data, including images of expected staining patterns .

• Species reactivity: Most TMEM31 antibodies show reactivity primarily with human samples, with limited cross-reactivity to other species .

• Conjugation: Consider whether native (unconjugated) or conjugated (FITC, HRP) antibodies better suit your experimental design .

• Epitope region: For membrane proteins like TMEM31, epitope accessibility in your experimental conditions is critical.

What validation strategies are essential before using TMEM31 antibodies in critical experiments?

Comprehensive validation should include:

• Positive and negative controls: Use testis tissue as a positive control and other normal tissues as negative controls .

• Concentration optimization: Perform titration experiments to determine optimal working concentration for your specific application.

• Specificity validation:

  • Western blot analysis showing expected molecular weight

  • Testing in TMEM31 knockdown/knockout models

  • Preabsorption with recombinant TMEM31 protein

• Comparative analysis: When possible, validate results with multiple antibodies targeting different TMEM31 epitopes.

• Method comparison: Correlate protein detection with mRNA expression data to confirm specificity.

For Immunohistochemistry:

• Fixation: Formalin-fixed paraffin-embedded (FFPE) samples with standard processing

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Blocking: 5-10% normal serum from secondary antibody host species

• Primary antibody: Dilute 1:20-1:200 in blocking buffer; incubate overnight at 4°C

• Detection system: HRP-conjugated secondary antibody or polymer detection system

• Counterstain: Hematoxylin for nuclear visualization

For Immunofluorescence:

• Fixation: 4% paraformaldehyde for 15-20 minutes

• Permeabilization: 0.1-0.2% Triton X-100 for 10 minutes

• Primary antibody: Dilute 1:50-1:200; incubate overnight at 4°C

• Secondary detection: Fluorophore-conjugated secondary antibodies or direct detection if using conjugated primary antibodies like TMEM31-FITC

• Counterstain: DAPI for nuclear visualization

For optimal TMEM31 detection in HepG2 cells, researchers have successfully used a 1:100 dilution with Alexa Fluor 488-conjugated secondary antibodies .

How can researchers troubleshoot common issues with TMEM31 antibody applications?

For storage and handling, TMEM31 antibodies should be aliquoted and stored at -20°C or -80°C to avoid repeated freeze-thaw cycles . For conjugated antibodies (FITC), protection from light is essential to prevent fluorophore degradation .

How can TMEM31 antibodies be utilized in cancer research, particularly for melanoma?

TMEM31's emerging role as a potential biomarker for melanoma progression offers several research applications:

• Expression profiling across tumor stages: Using IHC with TMEM31 antibodies, researchers have observed significantly higher expression in metastatic melanoma compared to primary melanoma (integrated optical density analysis: 0.24±0.03 vs. 0.09±0.01; p<0.001) .

• Metastasis mechanism investigation: Studying TMEM31's function in cell migration, invasion, and cellular signaling during metastatic progression.

• Patient stratification: Evaluating TMEM31 expression (76.56% in metastatic vs. 56.35% in primary melanoma; p=0.017) as a potential prognostic biomarker .

• Therapeutic target validation: Using antibodies to confirm target accessibility in tumor models prior to developing targeted therapies.

• Vaccine development research: TMEM31 has been identified as a target for multiepitope cancer vaccines against melanoma, with specific immunodominant fragments (residues 32-62, 77-105, and 125-165) selected for vaccine design .

What considerations apply when using TMEM31 antibodies in multiplexed detection systems?

When incorporating TMEM31 antibodies into multiplexed immunofluorescence or other multiparameter detection systems:

• Panel design: Combine with markers relevant to your research question:

  • For cancer research: Proliferation markers (Ki-67), cancer stem cell markers (CD44)

  • For subcellular localization: Membrane compartment markers

• Technical compatibility:

  • Antibody host species should be considered to avoid cross-reactivity

  • For spectral imaging, select fluorophores with minimal spectral overlap

  • If using multiple rabbit antibodies, consider sequential staining with stripping

• Validation requirements:

  • Validate each antibody individually before multiplexing

  • Compare multiplex results with single staining to ensure consistency

  • Include appropriate controls for each parameter

• Analysis methods:

  • Develop standardized quantification approaches

  • Consider automated image analysis for objective quantification

  • Validate results across multiple tissue sections or experimental replicates

How can TMEM31 antibody research be integrated with genetic manipulation technologies?

Combining antibody-based detection with genetic approaches provides complementary insights:

• CRISPR-Cas9 modification: TMEM31 guide RNAs designed by the Zhang laboratory at the Broad Institute are available for CRISPR-based gene editing . This enables:

  • Creating knockout cell lines for antibody validation

  • Studying phenotypic effects of TMEM31 depletion

  • Engineering reporter systems for live-cell imaging

• Overexpression systems: Transfecting cells with TMEM31 expression constructs to:

  • Create positive controls for antibody validation

  • Investigate effects of TMEM31 upregulation

  • Test structure-function relationships with mutated variants

• Multi-omics integration: Combining antibody-based protein detection with:

  • Transcriptomic analysis (RNA-seq, qPCR)

  • Epigenetic studies (ChIP-seq, methylation analysis)

  • Proteomic approaches (mass spectrometry, protein arrays)

What emerging antibody technologies might enhance TMEM31 research?

Recent developments in antibody engineering relevant to TMEM31 research include:

• Machine learning for antibody design: Computational approaches combining deep learning and multi-objective linear programming have shown promise for optimizing antibody properties . For TMEM31, this could enable:

  • Design of higher-affinity antibodies

  • Development of antibodies with novel specificities

  • Creation of cross-reactive antibodies for comparative species studies

• Affinity engineering approaches: Methods utilizing antibody repertoire data and ML for antibody affinity engineering have demonstrated success in recent studies . These approaches could:

  • Enhance detection sensitivity for low-abundance TMEM31

  • Improve specificity for particular TMEM31 epitopes or conformations

  • Enable discrimination between closely related protein family members

• Bispecific antibody development: Platforms for creating bispecific antibodies could be applied to TMEM31 research to:

  • Simultaneously target TMEM31 and immune effector cells

  • Enhance detection through dual epitope recognition

  • Develop therapeutic antibodies targeting TMEM31 in combination with other tumor markers

What are best practices for quantifying TMEM31 expression in tissue samples?

Robust quantification strategies include:

• Semi-quantitative scoring systems:

  • H-score (combines intensity and percentage of positive cells)

  • Allred score (combines proportion and intensity scores)

  • These systems should be validated through inter-observer reproducibility testing

• Digital image analysis:

  • Whole slide imaging with automated analysis algorithms

  • Standardized acquisition parameters (exposure, gain)

  • Appropriate segmentation for nuclear, cytoplasmic, and membrane staining

• Statistical considerations:

  • Sample size calculations based on expected effect size

  • Appropriate statistical tests for expression comparisons

  • Multi-variable analysis to account for confounding factors

• Reporting standards:

  • Document antibody details (manufacturer, clone, dilution)

  • Describe scoring methodology in detail

  • Include representative images spanning the scoring range

How should researchers evaluate conflicting results between TMEM31 antibody detection and other experimental methods?

When faced with discrepancies between antibody-based detection and other methods:

• Technical validation:

  • Verify antibody specificity through additional controls

  • Test multiple antibodies targeting different epitopes

  • Review fixation and antigen retrieval methods

• Biological considerations:

  • Evaluate post-translational modifications affecting antibody binding

  • Consider protein stability versus mRNA stability differences

  • Assess potential splice variants or protein isoforms

• Methodological resolution approaches:

  • Proximity ligation assays to confirm protein-protein interactions

  • Subcellular fractionation to resolve localization discrepancies

  • Correlation with functional assays to understand biological significance

• Integrated analysis:

  • Triangulate results using multiple complementary methods

  • Consider temporal factors in protein versus mRNA expression

  • Evaluate cell type-specific expression patterns in heterogeneous samples

What novel applications of TMEM31 antibodies are emerging in translational research?

Emerging translational applications include:

• Liquid biopsy development: Exploring TMEM31 detection in circulating tumor cells or extracellular vesicles for minimally invasive cancer monitoring.

• Theranostic approaches: Developing TMEM31 antibodies conjugated to both imaging agents and therapeutic payloads for simultaneous diagnosis and treatment.

• Immunotherapy applications: Creating TMEM31-targeted vaccines, CAR-T cells, or antibody-drug conjugates, leveraging its cancer/testis antigen status.

• Precision medicine stratification: Using TMEM31 expression patterns to guide therapy selection in metastatic melanoma or other TMEM31-expressing cancers.

What research gaps remain in understanding TMEM31 function and antibody applications?

Despite progress, several knowledge gaps persist:

• Functional characterization: Limited understanding of TMEM31's normal biological function and its role in cancer progression.

• Interacting partners: Few studies have identified proteins that interact with TMEM31 or signaling pathways it influences.

• Cross-reactivity: Limited validation of antibody specificity across closely related protein family members.

• Therapeutic potential: Early-stage exploration of TMEM31 as a therapeutic target, with need for validation in diverse preclinical models.

• Clinical correlation: Preliminary evidence linking TMEM31 expression to clinical outcomes requires validation in larger cohorts with diverse patient populations.