TMEM11 Antibody

What is TMEM11 and where is it localized in cells?

TMEM11 is a mitochondrial transmembrane protein that localizes to the outer mitochondrial membrane (OMM). While previous studies suggested inner membrane localization, recent evidence definitively places TMEM11 at the OMM. This localization has been confirmed through multiple experimental approaches including protease protection assays, where TMEM11 (like the known OMM protein TOMM20) is completely digested by proteinase K treatment of intact mitochondria, while inner membrane proteins like MIC60 remain protected unless the outer membrane is disrupted . Furthermore, proximity-based APEX labeling visualization through electron microscopy shows TMEM11 forms dark precipitates on the exterior of mitochondria, consistent with OMM localization . Super-resolution confocal microscopy reveals that TMEM11 appears more uniformly distributed along the mitochondrial membrane compared to inner membrane proteins like MIC60, which concentrate at discrete focal structures enriched at cristae junctions .

What experimental techniques require validated TMEM11 antibodies?

TMEM11 antibodies are essential for multiple experimental applications including:

• Western blot analysis to evaluate TMEM11 expression levels or knockdown efficiency

• Immunoprecipitation for studying protein-protein interactions

• Immunofluorescence microscopy to visualize TMEM11 localization

• Protease protection assays to determine membrane topology

• Blue Native PAGE (BN-PAGE) to analyze native protein complexes

Researchers should validate antibodies for specificity using knockout or knockdown controls. For example, CRISPR interference (CRISPRi) has been successfully used to generate TMEM11-depleted cell lines that exhibit nearly complete absence of TMEM11 as confirmed by Western analysis, providing excellent negative controls for antibody validation .

How can researchers distinguish between endogenous TMEM11 and tagged versions in experimental systems?

When simultaneously studying endogenous TMEM11 and exogenously expressed tagged versions, researchers should employ multiple detection strategies. Western blotting can reveal mobility shifts between native TMEM11 and tagged versions (GFP-TMEM11 or APEX2-GFP-TMEM11). In studies examining TMEM11 function, researchers have successfully restored normal mitochondrial morphology in TMEM11-depleted cells by expressing GFP- or APEX2-GFP-tagged TMEM11, confirming these tagged versions maintain functionality .

When performing co-localization studies, use antibodies targeting different epitopes or regions of TMEM11 in combination with antibodies against tag sequences. In quantitative analyses, employ densitometry to compare expression levels between endogenous and tagged proteins. Published studies have confirmed that GFP-tagged TMEM11 constructs typically exhibit modest overexpression compared to endogenous levels but completely alleviate morphological defects in depleted cells .

What are the recommended protocols for using TMEM11 antibodies in studying mitochondrial morphology?

To investigate TMEM11's role in mitochondrial morphology:

Protocol overview:

• Establish experimental and control conditions (TMEM11 knockdown vs. control cells)

• Stain mitochondria with vital dyes like Mitotracker

• Perform immunofluorescence with TMEM11 antibodies

• Acquire high-resolution images using confocal microscopy

• Quantify morphological parameters

TMEM11-depleted cells characteristically exhibit enlarged and/or bulbous mitochondria compared to the narrow tubular mitochondria in control cells. Published studies typically quantify this by categorizing mitochondrial morphology across multiple cells, with more than half of TMEM11-depleted cells showing altered morphology .

Image analysis recommendations:

• Use super-resolution techniques like SoRa confocal microscopy for detailed visualization

• Compare TMEM11 localization with other mitochondrial markers such as matrix-localized HSP60 or OMM markers like TOMM20

• Quantify mitochondrial width, length, and branching patterns

• Perform blind scoring of morphological categories by multiple observers

How should researchers optimize co-immunoprecipitation protocols with TMEM11 antibodies?

For successful co-immunoprecipitation (co-IP) of TMEM11 and its interaction partners:

Optimized co-IP protocol:

• Cell lysis: Use mild detergents that preserve membrane protein interactions

• Pre-clearing: Remove non-specific binding proteins with appropriate control beads

• Immunoprecipitation: Incubate lysates with anti-TMEM11 antibody coupled to Protein G beads

• Washing: Perform stringent washes to remove non-specific interactions

• Elution and analysis: SDS-PAGE followed by immunoblotting for potential interaction partners

Validated interaction partners:
TMEM11 robustly interacts with BNIP3 and BNIP3L as confirmed by reciprocal co-IP experiments . While interactions with VDAC1 can be detected, they appear less robust, and TMEM11 does not interact with the abundant OMM protein TOMM20, indicating specificity of the BNIP3/BNIP3L interactions .

For verification of interactions, researchers should perform reciprocal co-IPs (e.g., immunoprecipitate with anti-BNIP3 or anti-BNIP3L antibodies and probe for TMEM11) . Direct interaction can be further confirmed using yeast two-hybrid systems as demonstrated in published studies .

What approaches are recommended for troubleshooting non-specific binding with TMEM11 antibodies?

Common issues with TMEM11 antibodies include high background and non-specific binding. To address these challenges:

• Antibody validation: Confirm specificity using TMEM11 knockdown controls. CRISPR interference (CRISPRi) with sgRNAs targeting the TMEM11 transcription start site has been successful in creating nearly complete depletion of TMEM11 .

• Blocking optimization:

  • For Western blotting: Test different blocking solutions (BSA vs. milk) and concentrations

  • For immunofluorescence: Extended blocking times (1-2 hours) with serum matching secondary antibody host

• Antibody dilution series: Perform titration experiments to determine optimal concentrations that maximize specific signals while minimizing background.

• Secondary antibody controls: Include samples with secondary antibody only to identify non-specific binding.

• Cross-adsorbed secondary antibodies: Use highly cross-adsorbed secondary antibodies to minimize species cross-reactivity.

When analyzing proteomic data from TMEM11 immunoprecipitation, background subtraction of proteins identified in control samples is essential before assigning normalized spectral abundance factor (NSAF) scores to account for protein molecular weight differences .

What methodologies are recommended for studying TMEM11's interaction with the MICOS complex?

To investigate TMEM11's relationship with the MICOS (Mitochondrial Contact Site and Cristae Organizing System) complex:

Recommended approach:

• Co-immunoprecipitation with mass spectrometry: Perform IP with anti-TMEM11 antibodies followed by mass spectrometry-based proteomic analysis. Previous studies have successfully identified multiple MICOS complex components including MIC60 and MIC19 using this approach .

• Blue Native PAGE (BN-PAGE): Use 2D BN-PAGE to analyze native protein complexes. This technique has confirmed TMEM11's association with the MICOS complex .

• Super-resolution microscopy: Employ SoRa confocal microscopy to examine co-localization between TMEM11 and MICOS components like MIC60. Studies have shown that while MIC60 concentrates at discrete focal structures at cristae junctions, TMEM11 appears more uniformly distributed, suggesting functional roles independent of MICOS .

• Proximity labeling: Use techniques like BioID or APEX2 to identify spatial relationships between TMEM11 and MICOS components.

• Functional assays: Assess MICOS complex integrity and function in TMEM11-depleted cells by examining cristae morphology using electron microscopy.

How can TMEM11 antibodies be utilized to investigate its role in mitophagy?

TMEM11 co-enriches with mitophagy receptors BNIP3 and BNIP3L at mitophagy sites, and its absence leads to dysregulated mitophagy site formation . To investigate this function:

Experimental approach:

• Co-localization analysis:

  • Induce mitophagy using appropriate stimuli

  • Perform immunofluorescence with antibodies against TMEM11, BNIP3/BNIP3L, and mitophagy markers

  • Quantify co-localization at mitophagy sites using high-resolution microscopy

• Biochemical fractionation:

  • Isolate mitophagosomes using density gradient centrifugation

  • Analyze TMEM11 enrichment in these fractions by Western blotting

• Live-cell imaging:

  • Express fluorescently-tagged TMEM11 and mitophagy markers

  • Monitor temporal dynamics during mitophagy induction

  • Compare wild-type vs. TMEM11-depleted cells

• Functional assays:

  • Measure mitophagy flux in control vs. TMEM11-depleted cells

  • Quantify the number and distribution of mitophagy sites along mitochondrial membranes

  • Assess the impact of TMEM11 absence on BNIP3/BNIP3L recruitment

Research has shown that in the absence of TMEM11, mitophagy sites are dysregulated and dramatically increase in number along the mitochondrial membrane , suggesting TMEM11 plays a role in spatially restricting mitophagy initiation.

What techniques are available for studying TMEM11's role in cardiomyocyte proliferation?

TMEM11 has been identified as an inhibitor of cardiomyocyte proliferation and cardiac regeneration, with important implications for cardiac repair after injury . To investigate this function:

Experimental strategies:

• Genetic manipulation models:

  • TMEM11 knockout enhances cardiomyocyte proliferation and improves heart function after myocardial injury

  • TMEM11 overexpression inhibits neonatal cardiomyocyte proliferation and regeneration in mouse hearts

• Signaling pathway analysis:

  • TMEM11 directly interacts with METTL1 and enhances m7G methylation of Atf5 mRNA

  • This increases ATF5 expression, which promotes transcription of Inca1 (an inhibitor of cyclin-dependent kinase)

  • The TMEM11-METTL1-ATF5-INCA1 axis suppresses cardiomyocyte proliferation

• Quantitative assays:

  • Measure cardiomyocyte proliferation markers (Ki67, EdU incorporation, Aurora B)

  • Assess cardiac function using echocardiography

  • Analyze infarct size and ventricular remodeling after injury

• Molecular interaction studies:

  • Use TMEM11 antibodies for co-IP to confirm interactions with METTL1

  • Employ RNA immunoprecipitation to analyze m7G methylation of target transcripts

This research area suggests targeting the TMEM11-METTL1-ATF5-INCA1 axis may represent a novel therapeutic strategy for promoting cardiac repair and regeneration .

What methodological considerations apply when using TMEM11 antibodies with protein topology analysis techniques?

Determining the precise topology of TMEM11 in the outer mitochondrial membrane requires specialized approaches:

Protocol recommendations:

• Protease protection assays:

  • Isolate intact mitochondria by differential centrifugation

  • Treat with proteinase K before or after disruption of the outer membrane

  • Analyze by Western blotting with antibodies against TMEM11 and control proteins (e.g., TOMM20 for OMM, MIC60 for IMM)

• Carbonate extraction:

  • Isolate mitochondria and treat with carbonate solutions at varying pH (10.5, 11.5, or 12.5)

  • Centrifuge to separate peripheral membrane proteins (supernatant) from integral membrane proteins (pellet)

  • Analyze fractions by Western blotting

• Proximity labeling visualization:

  • Express APEX2-GFP-TMEM11 in cells

  • Treat with 3,3′-diaminobenzidine (DAB) and H₂O₂ post-fixation

  • Examine by electron microscopy to visualize TMEM11 localization relative to mitochondrial ultrastructure

• Fluorescent tag analysis:

  • Create constructs with fluorescent tags positioned at either N- or C-terminus

  • Express in cells and analyze subcellular localization

  • Determine which domains are exposed to cytosol vs. intermembrane space

Studies using these approaches have conclusively demonstrated that TMEM11 is an outer mitochondrial membrane protein, contrary to previous reports suggesting inner membrane localization .