tmem116 Antibody

What is TMEM116 and where is it expressed in normal tissues?

TMEM116 (Transmembrane Protein 116) is a member of the TMEM family of proteins that span cytoplasmic membranes and facilitate cell-cell and cell-environment communication. The function of this protein remains largely unknown . Expression studies have shown that TMEM116 is highly expressed in mouse kidney, lung, and heart tissues . During mouse lung embryonic development, TMEM116 expression dramatically increases from E16.5 and remains high until postnatal stages . At the cellular level, TMEM116 positive cells in lungs include airway Club cells, ciliated cells, basal cells, neuroendocrine cells, and alveolar type I and II cells .

Immunohistochemical analyses in human tissues have detected TMEM116 expression in:

• Testis (seminiferous duct cells)

• Gastrointestinal tissues (glandular cells)

• Stomach (glandular cells)

• Fallopian tube (glandular cells)

TMEM116 antibodies have been validated for various applications:

• Western Blotting (WB): Multiple antibodies have been validated for detecting TMEM116 protein expression levels in cell and tissue lysates .

• Immunohistochemistry (IHC): Several antibodies have been validated for detecting TMEM116 in formalin-fixed, paraffin-embedded tissues, with formic acid (FA) being identified as the most effective antigen retrieval method for some antibodies .

• Immunofluorescence (IF): Some antibodies have shown positivity in nuclear localization but not in nucleoli and cytoskeleton (microtubules) in human cell lines like A-431 .

• Immunoprecipitation (IP): Monoclonal antibodies like B-3 have been validated for pulling down TMEM116 protein complexes .

• ELISA: Some monoclonal antibodies have been validated for detection of TMEM116 in enzyme-linked immunosorbent assays .

How should TMEM116 antibodies be stored and handled for optimal performance?

Proper storage and handling of TMEM116 antibodies is critical for maintaining antibody integrity and experimental reproducibility:

Storage recommendations:

• For short-term use (up to 1 week): store at 2-8°C

• For long-term storage: store at -20°C in small aliquots to prevent freeze-thaw cycles

Buffer conditions:

• Most antibodies are supplied in PBS buffer with preservatives like 0.09% sodium azide and 2% sucrose

• Some are provided in lyophilized form and require reconstitution with distilled water

Handling precautions:

• Avoid repeated freeze-thaw cycles as they can denature the antibody and reduce activity

• Exercise caution with preservatives like sodium azide, which is classified as a poisonous and hazardous substance that should be handled by trained staff only

• Optimal working dilution should be determined empirically by each investigator

What is known about the protein structure and molecular characteristics of TMEM116?

TMEM116 is a transmembrane protein with the following characteristics:

• Protein size: 337 amino acids

• Molecular weight: Approximately 37 kDa

• Gene ID: 89894

• Transmembrane domains: As a member of the TMEM family, TMEM116 contains multiple membrane-spanning domains, though the exact topology has not been fully characterized

The C-terminal region of human TMEM116 contains the sequence "QRVRFYPVAFFCCWGPAVILMIIKLTKPQDTKLHMALYVLQALTATSQGL" (AA 229-278), which has been used as an immunogen for antibody production . Current research indicates that while the protein is expressed in various tissues, its precise function remains unknown .

What is known about TMEM116's role in cancer progression and metastasis?

Recent research has revealed significant insights into TMEM116's role in cancer:

TMEM116 expression is robustly increased in:

• Human lung cancer clinical samples

• Mouse lung cancer models

• Non-small-cell lung cancer (NSCLC) cell lines compared to normal human bronchial epithelial cells

Functional studies have demonstrated that:

• Inactivation of TMEM116 reduced cell proliferation, migration, and invasiveness of human cancer cells

• TMEM116 deficiency suppressed A549-induced tumor metastasis in mouse lungs

• TMEM116 knockdown inhibited PDK1-AKT-FOXO3A signaling pathway, resulting in accumulation of TAp63

Gene expression analysis revealed:

These findings suggest that TMEM116 functions as a critical integrator of oncogenic signaling in cancer metastasis, making it a potential therapeutic target for lung cancer treatment .

How does TMEM116 interact with signaling pathways in cancer cells?

Research has identified several key signaling pathways influenced by TMEM116:

PDK1-AKT-FOXO3A-TAp63 pathway:

• TMEM116 deficiency inhibits PDK1, phosphorylation of AKT and FOXO3A

• This inhibition activates TAp63 expression, which obstructs cell growth, migration, invasion, and metastasis

• Restoration of PDK1 by PS48 treatment recovers phosphorylation of AKT, blocks expression of TAp63, and restores cell growth, migration, and invasion in TMEM116-knockdown cells

AKT signaling specificity:

• Restoration of AKT phosphorylation by SC79 treatment only partially reverses TAp63 expression and cell migration/invasion

• Notably, it does not restore cell growth in TMEM116-knockdown cells

• This suggests that other proteins/effectors might be responsible for PDK1 signaling effects on cell growth

The data indicate that TMEM116 promotes cell migration and invasion partly through AKT/FOXO3A/TAp63 pathways, while its effects on proliferation may involve additional signaling mechanisms .

What methodologies are recommended for studying TMEM116 knockdown/knockout effects?

For effective TMEM116 functional studies, the following methodologies have been validated:

CRISPR-Cas9 gene editing:

• sgRNA targeting human TMEM116 has been effectively used in lung carcinoma cell lines

• Recommended transfection method: 2 μg of pCRISPR-W2-TMEM116 plasmids using Lipofectamine 2000

• Selection protocol: 48h post-transfection, culture cells in 800 μg/ml G418 for 1 week

• Example sgRNA sequences: sgRNA1: 5′-ACGGGTGCGCTTCTACCCAG-3′; sgRNA2: 5′-CATAAAGCTGACTAAGCCAC-3′

Functional assays for phenotype assessment:

• Colony formation assay: Assess changes in colony number, size, and morphology over 2 weeks

• Cell viability/proliferation: CCK8 assay to measure proliferation rates

• Migration assays: Wound-healing assay to evaluate cell motility

• Invasion assays: Transwell assays to measure invasive capacity

• In vivo metastasis model: Tail vein injection of control and TMEM116-knockdown cells into nude mice, followed by quantification of lung surface metastatic nodules

Validation of knockdown efficiency:

• Western blot analysis using validated anti-TMEM116 antibodies

• qRT-PCR for mRNA expression levels

CRISPR activation systems are also available for TMEM116 gene upregulation studies, using a SAM transcription activation system with D10A and N863A deactivated Cas9 fused to VP64 activation domain .

How can researchers address potential cross-reactivity issues with TMEM116 antibodies?

To address potential cross-reactivity issues with TMEM116 antibodies, researchers should implement several validation strategies:

Specificity validation methods:

• Adsorption tests: Pre-adsorb antibodies with peptide immunogens used for their generation (e.g., synthetic peptides corresponding to specific residues) to verify specific binding

• Knockout/knockdown controls: Use TMEM116 knockout or knockdown cell lines as negative controls for antibody validation

• Predicted cross-reactivity analysis: Consider predicted reactivity percentages with other species. For example, some antibodies show the following cross-reactivity: Human (100%), Cow (92%), Dog (93%), Guinea Pig (100%), Horse (93%), Rabbit (86%), Rat (100%)

• Multiple antibody approach: Use antibodies targeting different epitopes of TMEM116 to confirm expression patterns and localization. Compare results from different antibodies (e.g., N-terminal antibody vs. C-terminal antibody)

• Block peptide controls: Run parallel immunoassays with and without blocking peptides at determined concentrations (e.g., 30 μg of peptide immunogen)

When analyzing potential cross-reactivity, researchers should also consider sequence homology between TMEM116 and other TMEM family members, particularly in conserved domains.

What are the emerging therapeutic implications of targeting TMEM116 in cancer?

Research suggests several promising therapeutic implications for targeting TMEM116:

In lung cancer:

• TMEM116 is highly expressed in NSCLC tissues and cell lines

• TMEM116 inactivation reduces cell proliferation, migration, and invasion in vitro and suppresses metastasis in vivo

• This suggests TMEM116 could be a potential therapeutic target for lung cancer, particularly for preventing metastasis

Relationship with established cancer therapies:

• Research on other TMEM family members (TMEM16A/ANO1) has shown that their suppression improves response to antibody-mediated targeted cancer therapies

• TMEM16A inhibition in breast cancer cells with HER2 amplification induces loss of viability

• Cells resistant to trastuzumab (HER2-targeting antibody) showed increased TMEM16A expression

• Simultaneous cetuximab (EGFR antibody) treatment and TMEM16A suppression led to pronounced loss of viability in head and neck squamous cell carcinoma (HNSCC) cells

While these findings pertain to TMEM16A, they suggest that targeting TMEM family proteins, including TMEM116, might enhance the efficacy of existing targeted therapies through modulation of receptor tyrosine kinase signaling pathways. This represents an emerging area for combination therapy development .

What are the optimal immunohistochemistry protocols for TMEM116 detection in tissue samples?

Based on published research, the following optimized protocol is recommended for TMEM116 immunohistochemistry:

Tissue preparation:

• Fix tissues in 4% paraformaldehyde in PBS (pH 7.0)

• Process into serial paraffin sections (4-7 μm thickness)

Antigen retrieval:

• Formic acid (FA) treatment has been identified as the most effective antigen retrieval method for TMEM116 detection

• Recommended protocol: 1 minute FA treatment followed by washing in distilled water for 3 minutes

Blocking and primary antibody incubation:

• Deparaffinize and rehydrate sections

• Treat with 3% hydrogen peroxide in PBS for 30 minutes to eliminate endogenous peroxidase activity

• Block with protein block serum-free solution for 20 minutes

• Incubate with primary antibody overnight at 4°C

• Recommended antibody dilutions:

  • Anti-TMEM116 (residues 164-187): 1:50

  • Anti-TMEM116 (residues 188-211): 1:500

  • Anti-TMEM116 (residues 253-274): 1:500

  • Anti-TMEM116 (residues 239-250): 1:500

  • Anti-TMEM116 N-terminal: 1:1000

Detection and visualization:

• Wash in PBS

• Use an EnVision system (EnVision+ System-HRP-labeled Polymer)

• Visualize with diaminobenzidine (DAB)

• Counterstain with hematoxylin

Quantification:

What controls should be included in Western blotting experiments with TMEM116 antibodies?

For rigorous Western blotting with TMEM116 antibodies, the following controls should be included:

Positive controls:

• Cell lines with known TMEM116 expression (e.g., A549, H1299 lung cancer cell lines)

• Tissues with confirmed high expression (lung, kidney, heart)

• Recombinant TMEM116 protein (if available)

Negative controls:

• TMEM116 knockout or knockdown cell lines created using CRISPR-Cas9 or siRNA techniques

• Cell lines with low endogenous expression (e.g., 16HBE human bronchial epithelial cells have been shown to express lower levels of TMEM116 compared to cancer cell lines)

Technical controls:

• Loading control (β-actin, GAPDH, or another housekeeping protein)

• Molecular weight marker to confirm the expected 37 kDa band for TMEM116

• Secondary antibody-only control to assess non-specific binding

• Peptide competition assay: pre-incubate antibody with excess immunizing peptide to confirm specificity

Sample preparation considerations:

• Include both membrane and cytosolic fractions to account for potential processing of the transmembrane protein

• Consider using phosphatase inhibitors if studying phosphorylation-dependent interactions

• For cross-species studies, include samples from different species to validate predicted reactivity patterns

How can researchers optimize TMEM116 antibody dilutions for different applications?

Optimizing antibody dilutions is crucial for achieving specific signals while minimizing background. Here are application-specific recommendations:

Western Blotting (WB):

• Starting range: 1:500 to 1:2000

• Perform a titration series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Evaluate signal-to-noise ratio at each dilution

• Consider reducing primary antibody concentration if background is high

• Antibody concentration: Commercial TMEM116 antibodies are typically supplied at 0.76-1 mg/mL

Immunohistochemistry (IHC):

• Starting range: 1:50 to 1:500

• Different antibodies targeting different TMEM116 regions require different dilutions:

  • Anti-TMEM116 (residues 164-187): 1:50

  • Anti-TMEM116 (residues 188-211): 1:500

  • Anti-TMEM116 (residues 253-274): 1:500

  • Anti-TMEM116 (residues 239-250): 1:500

• Include positive and negative tissue controls at each dilution

• Evaluate staining pattern, intensity, and background

Immunofluorescence (IF):

• Starting range: 1-4 μg/mL or 1:100 to 1:500

• Titrate and evaluate signal intensity versus background autofluorescence

• Include a nuclear stain to evaluate localization patterns

General optimization principles:

• "Optimal working dilution should be determined by the investigator"

• Test multiple blocking reagents if non-specific binding persists

• Consider longer incubation times with more dilute antibody solutions to improve signal-to-noise ratio

• Document optimal conditions for reproducibility across experiments

What approaches should be used to validate TMEM116 subcellular localization findings?

To validate TMEM116 subcellular localization findings, researchers should employ multiple complementary approaches:

Multi-method validation:

• Co-localization studies: Use established markers for cellular compartments

  • Membrane markers (e.g., Na+/K+ ATPase, pan-cadherin)

  • Nuclear markers (e.g., DAPI, Hoechst)

  • Endoplasmic reticulum markers (e.g., calnexin, KDEL)

  • Golgi apparatus markers (e.g., GM130)

• Multiple antibodies approach: Use antibodies targeting different epitopes

  • Compare N-terminal and C-terminal antibodies

  • Use monoclonal and polyclonal antibodies independently

  • Available data shows nuclear localization patterns with some antibodies

• Subcellular fractionation: Biochemically separate cellular compartments

  • Prepare cytosolic, membrane, nuclear, and cytoskeletal fractions

  • Analyze TMEM116 distribution across fractions by Western blotting

  • Include fraction-specific markers as controls

• Advanced imaging techniques:

  • Super-resolution microscopy to resolve fine subcellular structures

  • Live-cell imaging with fluorescently tagged TMEM116

  • Electron microscopy with immunogold labeling for precise localization

Validation controls:

• Peptide competition assays to confirm antibody specificity

• TMEM116 knockout/knockdown cells as negative controls

• Overexpression systems with tagged TMEM116 (with caution regarding artifactual localization)

• Treatment with drugs that disrupt specific cellular compartments to assess localization changes

Considerations for transmembrane proteins:

• TMEM116, as a transmembrane protein, may undergo processing or trafficking

• Consider examining both mature and precursor forms

• Investigate potential membrane microdomain associations (lipid rafts)

• Assess localization changes under relevant stimuli or pathological conditions

How can researchers investigate TMEM116 interactions with other proteins?

To investigate TMEM116 protein-protein interactions, researchers should consider the following methodological approaches:

Co-immunoprecipitation (Co-IP):

• Use TMEM116 antibodies validated for immunoprecipitation (e.g., TMEM116 Antibody B-3)

• Include appropriate controls (IgG control, TMEM116 knockout cells)

• Consider cross-linking before lysis for transient interactions

• Analyze precipitates using mass spectrometry for unbiased interaction discovery

• Follow up with targeted Western blotting for specific interaction partners (e.g., components of PDK1-AKT-FOXO3A pathway)

Proximity-based labeling approaches:

• BioID or TurboID: Fuse TMEM116 with a biotin ligase to biotinylate proximal proteins

• APEX2: Fuse TMEM116 with an engineered peroxidase for proximity labeling

• These approaches can identify both stable and transient interactions in living cells

Förster Resonance Energy Transfer (FRET):

• Generate fluorescently tagged TMEM116 constructs

• Co-express with fluorescently tagged potential interaction partners

• Measure FRET efficiency to assess protein proximity (<10 nm)

• Particularly useful for membrane protein interactions

Yeast two-hybrid screening:

• Use TMEM116 domains (especially cytoplasmic domains) as bait

• Screen against cDNA libraries from relevant tissues

• Validate hits using other interaction methods

Split complementation assays:

• Bimolecular Fluorescence Complementation (BiFC)

• Split luciferase complementation

• These provide visual confirmation of protein interactions in live cells

Computational predictions and analysis:

• Use protein-protein interaction databases to identify potential interactors

• Validate computationally predicted interactions experimentally

• Consider known interactions of other TMEM family members as potential candidates

For the PDK1-AKT-FOXO3A-TAp63 pathway implicated in TMEM116 function , researchers should specifically investigate how TMEM116 interacts with these signaling components, potentially through direct or indirect mechanisms.