TMEM106A Antibody

What is TMEM106A and why is it significant for research?

TMEM106A (Transmembrane Protein 106A) is a type II transmembrane protein with a cytoplasmic region (amino acids 1-95), a transmembrane region (amino acids 96-115), and an extracellular region (amino acids 116-262) . It has emerged as a significant research target due to its multifaceted roles as a tumor suppressor in various cancers, particularly hepatocellular carcinoma (HCC), and as an antiviral factor that inhibits both enveloped and specific non-enveloped viruses . Additionally, TMEM106A expresses constitutively on macrophage plasma membranes where it regulates M1 polarization and pro-inflammatory functions . Its conservation across multiple species including humans, chimpanzees, mice, and rats suggests fundamental biological importance, making it a valuable target for antibody-based research applications .

What types of TMEM106A antibodies are available for laboratory research?

For TMEM106A research, researchers typically employ several categories of antibodies with distinct applications:

• Monoclonal antibodies: Provide high specificity for single epitopes, ideal for consistent results in applications like flow cytometry, immunoprecipitation, and Western blotting

• Polyclonal antibodies: Recognize multiple epitopes, enhancing detection sensitivity in applications like immunohistochemistry where protein conformation may vary

• Domain-specific antibodies: Target particular regions of TMEM106A, such as:

  • N-terminal (cytoplasmic domain) antibodies for intracellular detection

  • C-terminal (extracellular domain) antibodies for cell surface studies and blocking experiments

• Tag-specific antibodies: Used with tagged recombinant TMEM106A constructs in overexpression studies

Selection should be based on the experimental design, target localization, and whether native or denatured protein detection is required.

How should researchers validate TMEM106A antibody specificity for their experimental models?

A comprehensive validation strategy for TMEM106A antibodies should include multiple complementary approaches:

• Positive and negative control tissues/cells: Compare expression between:

  • Normal liver tissue (positive control - high TMEM106A expression)

  • HCC specimens (potential negative control - often shows downregulation)

  • TMEM106A-knockout cell lines generated via CRISPR-Cas9

• Peptide competition assays: Pre-incubate the antibody with increasing concentrations of the immunizing peptide before application to samples. Specific binding should be competitively eliminated.

• Orthogonal validation: Compare results with alternative detection methods:

  • RT-qPCR to correlate protein detection with mRNA expression

  • Multiple antibodies targeting different epitopes (results should align)

  • Mass spectrometry validation of immunoprecipitated proteins

• Knockdown/overexpression validation: Test antibody response in:

  • TMEM106A-knockdown cells (should show reduced signal)

  • TMEM106A-overexpressing cells (should show enhanced signal)

  • Perform controlled transfection experiments with TMEM106A constructs

Researchers should document all validation steps thoroughly in publications to establish antibody reliability.

What are the optimal fixation and antigen retrieval methods for TMEM106A immunohistochemistry?

Due to TMEM106A's membrane localization and potential conformation-dependent epitopes, careful optimization of sample preparation is crucial:

Fixation methods comparison:

Antigen retrieval optimization:

• For FFPE samples, heat-induced epitope retrieval (HIER) is generally effective:

  • Citrate buffer (pH 6.0) for 20 minutes is a good starting point

  • For challenging samples, try Tris-EDTA (pH 9.0)

  • Always include positive control tissues to confirm retrieval effectiveness

• Enzymatic retrieval may be necessary for some fixatives:

  • Proteinase K (10μg/mL for 10-15 minutes)

  • Control treatment time carefully to prevent over-digestion

• For dual staining protocols with other markers, select compatible retrieval methods or perform sequential staining with separate retrieval steps.

Testing a matrix of fixation and retrieval methods with control tissues is recommended before proceeding with valuable experimental samples .

How can TMEM106A antibodies be used to study its role in cancer suppression and EMT inhibition?

TMEM106A antibodies can provide crucial insights into cancer biology through multiple advanced applications:

These techniques collectively help delineate TMEM106A's role in tumor suppression and provide potential therapeutic insights for cancers with TMEM106A dysregulation .

What considerations are important when using TMEM106A antibodies to investigate viral interactions?

When designing experiments to study TMEM106A's antiviral functions, researchers should consider several important factors:

• Epitope selection and accessibility:

  • Antibodies targeting the extracellular region (amino acids 116-262) are crucial for studying interactions with viruses and SCARB2

  • The transmembrane-anchored extracellular region is essential for antiviral activity, suggesting membrane positioning is important

  • Non-blocking antibodies should be selected for co-localization studies to avoid interfering with the natural virus-protein interaction

• Virus-specific experimental design:

  • For enveloped viruses (like HIV-1): Focus on virion release inhibition assays, as TMEM106A traps viral particles at the cell surface

  • For non-enveloped viruses (like EV-A71 and CV-A16): Design binding competition assays between virus and SCARB2 in the presence of TMEM106A

  • Controls should include CV-A10, which is not affected by TMEM106A expression

• Temporal considerations:

  • Design time-course experiments with synchronized infection to determine if TMEM106A acts at early attachment stages or later release stages

  • For type I interferon-induced expression studies, optimize pre-treatment timing based on TMEM106A induction kinetics

• Co-localization analysis:

  • Use subcellular fractionation followed by Western blotting to confirm membrane localization

  • For advanced analysis, consider super-resolution microscopy techniques like STORM or PALM to precisely map TMEM106A-virus interactions at the nanoscale

  • Include appropriate controls for antibody specificity in co-localization studies

• Functional blocking experiments:

  • Compare the effects of antibodies targeting different domains of TMEM106A on viral infection rates

  • Include Fab fragments to distinguish between steric hindrance and specific functional blocking

These methodological considerations are essential for accurately interpreting TMEM106A's role in viral restriction mechanisms .

How can confocal microscopy be optimized for TMEM106A antibody-based detection of protein-protein interactions?

Confocal microscopy with TMEM106A antibodies requires careful optimization to reveal meaningful protein-protein interactions:

• Sample preparation refinements:

  • For membrane protein preservation, gentle fixation (2% PFA for 10-15 minutes) is preferable to harsher methods

  • Consider detergent selection carefully: 0.1% Triton X-100 for general permeabilization, but 0.01% saponin may better preserve membrane structures

  • For co-localization with SCARB2, optimize sequential staining protocols if antibody species overlap occurs

• Advanced co-localization analysis techniques:

  • Beyond simple overlay, employ quantitative co-localization metrics:

    • Pearson's correlation coefficient (measures linear relationships)

    • Manders' overlap coefficient (proportion of overlapping signals)

    • Object-based co-localization (for punctate structures)

  • Create intensity correlation plots to visualize co-dependency of fluorescence intensities

• Specialized techniques for membrane protein interactions:

  • FRET (Fluorescence Resonance Energy Transfer) microscopy can detect direct interactions (<10nm) between TMEM106A and binding partners

  • TIRF (Total Internal Reflection Fluorescence) microscopy offers superior resolution at the plasma membrane where TMEM106A functions

  • Implement deconvolution algorithms to enhance signal resolution for membrane proteins

• Controls for specificity and quantification:

  • Use structured illumination to enhance resolution beyond the diffraction limit

  • Include sample transfected with different truncated forms of TMEM106A (a.a 1-120, a.a 1-170, etc.) as shown in the literature

  • For interaction with SCARB2, include controls with antibodies directed against different regions (helices 2, 5, and 14) to map interaction domains

• Data analysis recommendations:

  • Employ masked analysis protocols where the investigator is blinded to sample identity

  • Use automated threshold determination to avoid subjective bias in co-localization assessment

  • Consider machine learning approaches for pattern recognition in complex co-localization studies

These optimizations enable precise visualization of TMEM106A interactions with partners like SCARB2 and viral particles at subcellular resolution .

How can researchers address background signal issues when using TMEM106A antibodies in immunostaining?

Background signal challenges with TMEM106A antibodies can be systematically addressed:

• Common sources of background and targeted solutions:

• Tissue-specific optimizations for TMEM106A detection:

  • For liver tissue (where TMEM106A expression varies between normal and HCC): Include longer blocking steps and implement Sudan Black B treatment to reduce autofluorescence

  • For macrophage studies: Add a specific Fc receptor blocking step before antibody incubation, as TMEM106A is constitutively expressed on macrophage plasma membranes

• Antibody dilution optimization protocol:

  • Perform a systematic dilution series (starting from 1:100 to 1:5000)

  • Score signal-to-noise ratio at each dilution

  • The optimal dilution should provide clear positive signal with minimal background

  • For low-abundance samples, consider signal amplification systems (tyramide signal amplification) rather than less-dilute primary antibody

• Advanced clearing techniques for thick tissue sections:

  • For 3D visualization of TMEM106A in tissue context, implement tissue clearing methods (CLARITY, CUBIC, or iDISCO)

  • Optimize antibody penetration with extended incubation periods (48-72 hours) and gentle agitation

  • Include detergent titration to balance membrane preservation with antibody accessibility

These strategies will help maximize signal specificity while minimizing background interference in TMEM106A detection .

What are the optimal conditions for using TMEM106A antibodies in co-immunoprecipitation experiments?

Successful co-immunoprecipitation (co-IP) of TMEM106A with its interaction partners requires careful protocol optimization:

• Lysis buffer optimization for membrane protein extraction:

  • Test multiple buffer compositions:

    • Standard RIPA buffer often disrupts membrane protein interactions

    • Milder NP-40 buffer (1% NP-40, 150mM NaCl, 50mM Tris pH 8.0) preserves more interactions

    • For studying TMEM106A-SCARB2 interactions, consider digitonin-based buffers (1% digitonin) which better maintain membrane protein complexes

  • Include protease inhibitors, phosphatase inhibitors, and EDTA to prevent degradation

• IP strategy selection based on research goals:

  • Direct IP: Use anti-TMEM106A antibodies directly conjugated to beads (reduces heavy chain interference in subsequent Western blots)

  • Reverse IP: Immunoprecipitate suspected binding partners (SCARB2, components of Erk1/2/Slug pathway) and probe for TMEM106A

  • For weak/transient interactions: Consider crosslinking approaches (DSP, formaldehyde) prior to lysis

  • For difficult membrane protein complexes: Try proximity-based labeling (BioID, APEX) as an alternative to traditional co-IP

• Technical refinements to improve success rate:

  • Pre-clear lysates thoroughly (1 hour with protein A/G beads) to reduce non-specific binding

  • Optimize antibody amounts: Typically 2-5μg antibody per 500μg-1mg protein lysate

  • Extended incubation at 4°C (overnight) with gentle rotation improves capture efficiency

  • For washing, use decreasing salt concentration series to gradually reduce stringency

• Controls and validation approaches:

  • Include isotype control antibodies matching the TMEM106A antibody class and species

  • Use knockout/knockdown samples as negative controls

  • For suspected interactions (e.g., with SCARB2), include competition with recombinant protein

  • Consider size exclusion chromatography as an orthogonal approach to validate interactions

• Detection optimization:

  • For Western blot detection after IP, use HRP-conjugated protein A/G instead of secondary antibodies to reduce background

  • Consider specialized gel systems for membrane proteins (Tricine-SDS-PAGE)

  • For weak interactions, employ highly sensitive detection methods (ECL Advance, Clarity Max)

These optimized approaches facilitate detection of both constitutive and transient interactions of TMEM106A with its biologically relevant partners .

How can researchers quantitatively assess TMEM106A expression levels across different experimental conditions?

Quantitative assessment of TMEM106A expression requires rigorous methodology and appropriate controls:

• Western blot quantification refinements:

  • Include a concentration gradient of recombinant TMEM106A to establish a standard curve

  • Normalize to multiple housekeeping proteins (β-actin, GAPDH, α-tubulin) for robust comparison

  • For membrane protein normalization, consider Na⁺/K⁺-ATPase or calnexin as more appropriate references

  • Implement technical triplicates and multiple biological replicates (minimum n=3)

  • Use fluorescence-based detection systems (LI-COR) for wider linear detection range compared to chemiluminescence

• Flow cytometry protocol for cell surface TMEM106A quantification:

  • Optimize gentle cell dissociation to preserve membrane epitopes (enzyme-free dissociation buffers)

  • Use quantitative flow cytometry with calibration beads (Quantum Simply Cellular beads) to determine Antibody Binding Capacity (ABC)

  • Include compensation controls when performing multicolor analysis with other markers

  • For intracellular epitopes, compare different permeabilization methods (saponin vs. Triton X-100)

• RT-qPCR correlation with protein expression:

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Include multiple reference genes identified by stability algorithms (geNorm, NormFinder)

  • Correlate mRNA expression with protein levels to identify post-transcriptional regulation

  • For methylation studies, parallel analysis of TMEM106A promoter methylation and mRNA expression provides mechanistic insights

• Advanced quantitative imaging approaches:

  • Automated high-content imaging with standardized acquisition parameters

  • Machine learning-based segmentation for objective quantification of membrane vs. cytoplasmic localization

  • Implement FRET-based reporters to monitor dynamic changes in TMEM106A interactions

  • Use superresolution techniques (STED, STORM) for nanoscale quantification of clustering

• Integrated multi-omics approach:

  • Combine proteomics data with transcriptomics and epigenomics (methylation) for comprehensive expression profiling

  • Consider proteomic approaches like Selected Reaction Monitoring (SRM) for absolute quantification

  • For clinical samples, correlate TMEM106A levels with patient outcomes using appropriate statistical methods

Data normalization strategy:

These quantitative approaches enable robust comparison of TMEM106A levels across experimental conditions and clinical samples .

How can TMEM106A antibodies be used to study its role in hepatocellular carcinoma progression?

TMEM106A antibodies offer multiple approaches to investigate its tumor suppressor function in HCC:

These approaches collectively provide comprehensive insights into TMEM106A's role in HCC pathogenesis and potential therapeutic implications .

What experimental approaches can validate TMEM106A's antiviral mechanisms using specific antibodies?

To elucidate TMEM106A's antiviral functions, researchers can implement several antibody-based experimental strategies:

• Binding interference assays:

  • Use domain-specific antibodies to map critical regions for antiviral activity:

    • Compare antibodies targeting different regions of the extracellular domain (a.a. 116-262)

    • Correlate binding inhibition with antiviral effects

  • For SCARB2-mediated infections, perform competitive binding assays:

    • Pre-treat cells with anti-TMEM106A antibodies before virus attachment

    • Quantify virus binding efficiency through immunofluorescence or qPCR

    • Compare effects on susceptible viruses (EV-A71, CV-A16) vs. non-susceptible ones (CV-A10)

• Antibody-based virus attachment visualization:

  • Implement triple-labeling confocal microscopy:

    • Fluorescently labeled virus particles

    • Anti-TMEM106A antibodies (different fluorophore)

    • Anti-SCARB2 antibodies (third fluorophore)

  • Analyze co-localization patterns at cell surface

  • Include time-course imaging to capture dynamic attachment processes

  • Quantify co-localization coefficients under different conditions

• Structure-function relationship studies:

  • Use antibodies recognizing specific SCARB2 helices (2, 5, and 14) to map TMEM106A binding sites

  • Implement antibody accessibility assays:

    • Test whether TMEM106A expression blocks antibody binding to specific SCARB2 epitopes

    • Generate competitive binding profiles for different domains

  • Create domain-specific TMEM106A constructs and test antibody reactivity to map functional regions

• Type I interferon response monitoring:

  • Track TMEM106A upregulation following interferon treatment using quantitative immunofluorescence

  • Correlate TMEM106A induction with antiviral activity

  • Implement siRNA knockdown of interferon pathway components to map regulatory mechanisms

  • Use CRISPR-edited cells lacking interferon receptors as controls

• Combined therapeutic approach evaluation:

  • Test combinations of TMEM106A-inducing compounds with conventional antivirals

  • Use antibodies to monitor expression levels and localization changes

  • Quantify antiviral effects using plaque reduction assays or viral load measurements

  • Develop screening platforms for compounds that enhance TMEM106A expression or function

These experimental approaches provide comprehensive insights into TMEM106A's role in viral restriction, particularly focusing on its interactions with SCARB2 and mechanisms of action against both enveloped and non-enveloped viruses .

How can TMEM106A antibody-based assays help identify patients likely to respond to epigenetic therapy in cancer treatment?

TMEM106A methylation status and protein expression offer potential biomarker applications for epigenetic therapy response prediction:

Proposed decision matrix for patient stratification:

• Therapeutic decision guidance:

  • For patients with TMEM106A hypermethylation and poor prognosis :

    • Consider higher intensity demethylating therapy

    • More frequent monitoring of TMEM106A re-expression

    • Combination with pathway-specific therapies targeting Erk1/2

  • For patients without TMEM106A methylation:

    • Focus on alternative therapeutic targets

    • Consider combination approaches not dependent on epigenetic reactivation

This comprehensive approach leverages TMEM106A's epigenetic regulation to guide precision medicine approaches for cancer treatment, particularly in HCC where TMEM106A methylation correlates with clinical outcomes .

What novel TMEM106A antibody-based approaches could improve understanding of its dual role in cancer and antiviral immunity?

Innovative antibody-based strategies can bridge the gap between TMEM106A's seemingly disparate functions:

• Engineered antibody tools for mechanistic dissection:

  • Develop conformation-specific antibodies that distinguish between different functional states of TMEM106A

  • Create bispecific antibodies targeting TMEM106A and key interaction partners (SCARB2, components of Erk1/2 pathway)

  • Design activating/inhibitory antibodies that can modulate TMEM106A function for mechanistic studies

  • Implement optogenetic or chemically-inducible antibody fragments for temporal control of TMEM106A interactions

• Systems biology integration approaches:

  • Design multiplexed immunofluorescence panels incorporating:

    • TMEM106A expression

    • EMT markers (E-cadherin, N-cadherin, Vimentin)

    • Viral infection markers

    • Signaling pathway components (phospho-Erk1/2, Slug)

  • Apply spatial transcriptomics alongside protein detection for comprehensive molecular context

  • Implement high-dimensional analysis (t-SNE, UMAP) to identify cell state transitions associated with TMEM106A function

• Cross-disciplinary investigation opportunities:

  • Explore potential connections between TMEM106A's tumor suppressor and antiviral functions:

    • Does viral infection alter TMEM106A methylation status?

    • Can TMEM106A-mediated antiviral responses influence EMT processes?

    • Are cancer cells with TMEM106A silencing more susceptible to specific viral infections?

  • Design dual-purpose assays measuring both antitumor and antiviral functions simultaneously

• Translational development pathways:

  • Design theranostic approaches using TMEM106A antibodies:

    • Diagnostic imaging with radiolabeled antibodies to quantify protein expression

    • Therapeutic potential through antibody-drug conjugates targeting cells with aberrant TMEM106A expression

  • Develop antibody-based screens for compounds that:

    • Reverse TMEM106A methylation

    • Enhance TMEM106A antiviral activity

    • Modulate TMEM106A-dependent EMT regulation

• Innovative technological applications:

  • Implement CyTOF (mass cytometry) with TMEM106A antibodies for high-parameter analysis of protein networks

  • Apply super-resolution microscopy for nanoscale mapping of TMEM106A organization in membrane microdomains

  • Develop antibody-based proximity proteomics approaches (BioID, APEX) to comprehensively map TMEM106A interaction networks

  • Create organoid systems with reporter-linked TMEM106A antibodies for live monitoring of expression dynamics

These innovative approaches could reveal unexpected connections between TMEM106A's roles in cancer suppression via EMT inhibition and its antiviral functions, potentially identifying unified mechanisms and new therapeutic opportunities .

How might new single-cell technologies incorporating TMEM106A antibodies advance our understanding of its biology?

Single-cell technologies offer unprecedented opportunities to elucidate TMEM106A biology:

• Single-cell protein and transcriptome analysis:

  • Implement CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing):

    • Conjugate TMEM106A antibodies with DNA barcodes

    • Simultaneously capture protein expression and transcriptome

    • Correlate TMEM106A protein levels with gene expression programs

    • Identify cell subpopulations with unique TMEM106A functional states

  • Apply scATAC-seq in parallel to map chromatin accessibility at the TMEM106A locus across cell types

• High-dimensional cytometry approaches:

  • Design TMEM106A-focused CyTOF (mass cytometry) panels:

    • Include antibodies for TMEM106A, EMT markers, viral entry receptors

    • Add signaling pathway components (Erk1/2/Slug pathway)

    • Include epigenetic markers (e.g., H3K27me3)

  • Implement trajectory analysis to map cellular transitions associated with TMEM106A expression changes

  • Create visualization tools for multidimensional TMEM106A functional states

• Spatial biology integration:

  • Apply multiplexed ion beam imaging (MIBI) or CO-Detection by indEXing (CODEX):

    • Map TMEM106A expression in tissue context with subcellular resolution

    • Correlate with microenvironmental features

    • Identify spatial relationships with immune cells in tumor microenvironment

  • Implement cyclic immunofluorescence for high-parameter spatial mapping

  • Correlate spatial patterns with clinical outcomes in patient samples

• Live-cell dynamics and interaction monitoring:

  • Develop nanobody-based imaging tools for TMEM106A:

    • Create non-interfering labels for live imaging

    • Monitor dynamic reorganization during viral infection

    • Track interaction with SCARB2 and other binding partners

  • Apply optogenetic approaches to manipulate TMEM106A function with spatial precision

  • Implement FRET sensors to detect conformational changes upon ligand binding

• Innovative functional single-cell assays:

  • Single-cell secretome analysis coupled with TMEM106A expression profiling

  • Microfluidic platforms to correlate TMEM106A levels with cell migration and invasion at single-cell resolution

  • Combined viral infection and cell fate mapping at single-cell level

  • CRISPR perturbation screens with single-cell readouts of TMEM106A function

Proposed workflow for integrated single-cell TMEM106A analysis:

• Tissue dissociation with optimized protocols to preserve membrane proteins

• TMEM106A antibody labeling with DNA barcodes or metal isotopes

• Single-cell multi-omic analysis (protein, RNA, chromatin accessibility)

• Computational integration and trajectory mapping

• Functional validation of identified cell states using sorted populations

These advanced single-cell approaches would reveal heterogeneity in TMEM106A expression, regulation, and function across cell types and disease states, providing unprecedented insights into its biology .

What methodological considerations are important for developing TMEM106A-based therapeutic antibodies or diagnostics?

Developing TMEM106A-targeted diagnostics and therapeutics requires systematic consideration of key methodological factors:

• Diagnostic antibody development strategy:

  • Epitope selection considerations:

    • Target regions with consistent accessibility across tissue preparation methods

    • Select epitopes preserved in formalin-fixed tissues for clinical compatibility

    • Avoid regions subject to post-translational modifications that might affect recognition

  • Validation requirements for clinical diagnostics:

    • Extensive cross-reactivity testing against related TMEM family proteins

    • Reproducibility assessment across multiple antibody lots

    • Standardization of staining protocols for multi-center consistency

    • Correlation with orthogonal detection methods (qPCR, methylation analysis)

• Companion diagnostic development pathway:

  • Design standardized IHC scoring system for TMEM106A in tissue samples

  • Establish clinical cutoff values through ROC analysis with outcome data

  • Create decision algorithms integrating:

    • TMEM106A protein expression

    • Promoter methylation status

    • Clinical variables (tumor size, stage)

  • Develop quality control procedures for clinical laboratory implementation

• Therapeutic antibody engineering considerations:

  • For restoring TMEM106A function in tumors:

    • Non-blocking antibodies that enhance protein stability or trafficking

    • Bispecific antibodies linking TMEM106A to functional signaling components

  • For enhancing antiviral activity:

    • Antibodies that stabilize TMEM106A-SCARB2 interactions

    • Conformation-specific antibodies that promote antiviral state

  • Delivery strategies for membrane protein targeting:

    • Nanoparticle formulations for enhanced tumor penetration

    • Cell-type specific targeting to reduce off-target effects

• Methodological approaches for specificity enhancement:

  • Negative selection strategies against related TMEM family proteins

  • Affinity maturation through directed evolution approaches

  • Structure-guided optimization based on epitope mapping

  • Cross-species conservation analysis to identify functionally critical regions

• Translational development considerations:

  • Patient stratification based on TMEM106A status:

    • Methylation level

    • Protein expression pattern

    • Mutation/variant analysis

  • Combination therapy strategies:

    • With demethylating agents for tumors with TMEM106A silencing

    • With signaling pathway inhibitors (ERK inhibitors) for enhanced activity

    • With conventional antivirals for enhanced restriction of viral replication

Critical quality attributes for TMEM106A-targeted antibodies:

These methodological considerations provide a framework for developing TMEM106A-based clinical applications that leverage its roles in both cancer suppression and antiviral defense .