TMEM11 Antibody, HRP conjugated

What is TMEM11 and what cellular functions does it serve?

TMEM11 (Transmembrane protein 11, mitochondrial) is a protein localized to the outer mitochondrial membrane (OMM) that plays a critical role in mitochondrial morphogenesis. Research indicates that TMEM11 is distributed relatively uniformly along the mitochondrial membrane, occasionally localizing to discrete focal structures that appear distinct from MIC60-enriched cristae junctions. TMEM11 depletion via CRISPR interference (CRISPRi) in U2OS cells leads to observable changes in mitochondrial morphology, highlighting its importance in maintaining proper mitochondrial structure . Functionally, TMEM11 interacts with multiple mitochondrial proteins, including BNIP3 and BNIP3L, suggesting involvement in mitochondrial dynamics beyond simple structural maintenance .

What are the optimal storage conditions for TMEM11 Antibody, HRP conjugated?

TMEM11 Antibody, HRP conjugated should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be strictly avoided to maintain antibody integrity and activity. The typical storage buffer consists of 50% glycerol with 0.01M PBS (pH 7.4) containing 0.03% Proclin 300 as a preservative . For long-term storage projects, dividing the antibody into single-use aliquots before freezing is recommended to prevent activity loss from multiple freeze-thaw cycles.

What applications has the TMEM11 Antibody, HRP conjugated been validated for?

According to product specifications, commercially available TMEM11 Antibody, HRP conjugated has been specifically validated for Enzyme-Linked Immunosorbent Assay (ELISA) applications . While the primary validation is for ELISA, researchers have employed similar antibodies in Western blot analysis for detecting TMEM11 in studies investigating mitochondrial morphology and protein-protein interactions, particularly following immunoprecipitation procedures .

What is the immunogen used to generate commercially available TMEM11 antibodies?

The commercially available TMEM11 Antibody, HRP conjugated is typically generated using a recombinant fragment of human TMEM11 protein, specifically amino acids 1-83 . This region represents approximately the N-terminal portion of the TMEM11 protein, which may contain epitopes accessible for antibody binding. Understanding the specific immunogen is crucial when selecting antibodies for particular experimental approaches, especially when studying protein-protein interactions or potential post-translational modifications.

How should researchers design experiments to investigate TMEM11's role in mitochondrial dynamics?

When investigating TMEM11's role in mitochondrial dynamics, researchers should implement a multi-faceted experimental approach:

• Gene Knockdown/Knockout Systems: Utilize CRISPR interference (CRISPRi) or CRISPR-Cas9 technology to generate stable TMEM11 knockdown or knockout cell lines. For transient effects, siRNA can be employed. Confirm knockdown/knockout efficiency through both RT-qPCR and Western blot analysis .

• Microscopy Analysis: Employ vital dyes such as Mitotracker followed by fluorescence microscopy to examine mitochondrial morphology changes. Super-resolution techniques like SoRa confocal microscopy provide enhanced visualization of mitochondrial structures .

• Rescue Experiments: Perform complementation studies with GFP-tagged or APEX2-GFP-tagged TMEM11 constructs to confirm that observed phenotypes are specifically due to TMEM11 depletion rather than off-target effects .

• Co-localization Studies: Use immunofluorescence with markers for different mitochondrial compartments (e.g., TOMM20 for OMM, MIC60 for cristae junctions, HSP60 for matrix) to determine precise TMEM11 localization and potential redistribution under different conditions .

• Electron Microscopy: Implement proximity-based APEX labeling to visualize TMEM11 localization relative to mitochondrial ultrastructure using techniques like DAB staining with H₂O₂ .

What approaches are recommended for studying TMEM11's interaction with MICOS complex proteins?

To effectively study TMEM11's interactions with MICOS complex proteins, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Perform immunoprecipitation with anti-TMEM11 antibodies followed by Western blotting for MICOS components (MIC60, MIC19) or reverse Co-IP with MICOS antibodies followed by TMEM11 detection .

• Mass Spectrometry: Conduct immunoprecipitation coupled with mass spectrometry-based proteomic analysis to identify interaction partners. This approach should include proper controls and calculation of normalized spectral abundance factor (NSAF) scores to account for protein molecular weight .

• Blue Native PAGE (BN-PAGE): Implement 2D BN-PAGE analysis to study the interaction of TMEM11 with MICOS complex components under native conditions, helping preserve physiologically relevant protein complexes .

• Proximity Labeling: Apply techniques like BioID or APEX2 to identify proteins in close proximity to TMEM11 in living cells, which can reveal both stable and transient interactions.

• Yeast Two-Hybrid (Y2H) Analysis: Recapitulate interactome data by expressing constructs in a Y2H system to determine if TMEM11 can directly interact with MICOS components or if the interactions are indirect .

• Fluorescence Resonance Energy Transfer (FRET): Employ FRET analysis with fluorescently tagged TMEM11 and MICOS components to examine interactions in living cells.

What controls should researchers include when using TMEM11 Antibody, HRP conjugated in ELISA assays?

When conducting ELISA assays with TMEM11 Antibody, HRP conjugated, implementing proper controls is essential for result validation:

• Positive Control: Include recombinant TMEM11 protein at known concentrations to generate a standard curve and verify antibody functionality.

• Negative Control: Use samples known to lack TMEM11 expression or samples from TMEM11 knockout cell lines .

• Isotype Control: Include a non-specific rabbit IgG-HRP at equivalent concentration to assess non-specific binding .

• Blocking Optimization: Test different blocking reagents (BSA, non-fat milk, commercial blockers) to minimize background while maintaining specific signal.

• Antibody Titration: Perform a titration of the TMEM11 antibody to determine optimal working concentration that maximizes signal-to-noise ratio.

• Sample Matrix Control: Run buffer-only samples to establish baseline signal and detect potential interference from sample components.

• Cross-Reactivity Assessment: When working with non-human samples, validate species cross-reactivity using positive controls from the species of interest.

How can researchers optimize subcellular fractionation protocols for studying TMEM11 in mitochondrial membranes?

Optimizing subcellular fractionation for TMEM11 analysis requires careful attention to mitochondrial membrane preservation:

• Differential Centrifugation Protocol:

  • Homogenize cells in isotonic buffer (250 mM sucrose, 10 mM HEPES, pH 7.4, 1 mM EDTA)

  • Remove nuclei and debris with low-speed centrifugation (1,000g, 10 min)

  • Isolate crude mitochondria with medium-speed centrifugation (10,000g, 10 min)

  • Purify mitochondria further using sucrose gradient ultracentrifugation

• Membrane Separation: After isolating intact mitochondria, separate outer and inner membranes using hypotonic swelling followed by sonication and density gradient centrifugation .

• Alkaline Carbonate Extraction: Determine the membrane association strength of TMEM11 by treating mitochondrial fractions with Na₂CO₃ (pH 10-12.5) to distinguish peripheral from integral membrane proteins .

• Protease Protection Assays: Treat intact and permeabilized mitochondria with proteases to determine TMEM11 topology and membrane orientation.

• Quality Control Markers: Always validate fractionation quality using established markers: TOMM20 for outer membrane, cytochrome c for intermembrane space, TIM23 for inner membrane, and HSP60 for matrix .

• Western Blot Analysis: Analyze equal protein amounts from each fraction, using antibodies against compartment-specific markers alongside TMEM11 antibody.

What are the technical considerations for using TMEM11 antibodies in co-immunoprecipitation experiments?

When performing co-immunoprecipitation (Co-IP) with TMEM11 antibodies, these technical considerations are critical:

• Lysis Buffer Optimization: Use mild detergents (0.5-1% NP-40, CHAPS, or digitonin) that preserve protein-protein interactions while efficiently solubilizing membrane proteins. Avoid harsh detergents like SDS that disrupt protein complexes .

• Cross-linking Option: Consider implementing reversible cross-linking (e.g., DSP, formaldehyde) before lysis to capture transient or weak interactions, particularly important for membrane protein complexes.

• Pre-clearing Step: Pre-clear lysates with protein G beads to reduce non-specific binding.

• Antibody Controls: Include isotype-matched control antibodies and perform reverse Co-IPs (using antibodies against suspected interaction partners) to validate interactions .

• Elution Conditions: For FLAG-tagged constructs, use competitive elution with FLAG peptide (2 mg/ml) rather than harsh denaturing conditions to preserve interacting proteins .

• Detection Strategy: When blotting for interaction partners, optimize primary antibody concentrations and incubation conditions for each target protein .

• Validation Approaches: Confirm interactions through alternative methods such as proximity labeling, yeast two-hybrid, or in vitro binding assays .

How should researchers troubleshoot inconsistent results when detecting TMEM11 in different cell types?

When encountering inconsistent TMEM11 detection across different cell types, systematic troubleshooting is essential:

• Expression Level Verification: Confirm TMEM11 expression levels in different cell types via RT-qPCR before protein-level experiments, as expression can vary significantly between tissues and cell lines.

• Extraction Protocol Optimization: Adjust lysis conditions based on cell type; some cells may require stronger detergents or mechanical disruption to efficiently extract membrane proteins .

• Antibody Validation: Verify antibody specificity using TMEM11 knockdown or knockout samples as negative controls. Consider using multiple antibodies targeting different epitopes .

• Loading Control Selection: Choose appropriate loading controls specific for mitochondrial content (e.g., VDAC, TOM20) rather than general housekeeping proteins when comparing TMEM11 levels between cell types .

• Sample Preparation: For membrane proteins like TMEM11, avoid excessive heating during sample preparation, which can cause aggregation. Heat samples at lower temperatures (70°C instead of 98°C) or use alternative sample preparation methods.

• Detection Method Adjustment: Optimize primary antibody concentration and incubation time for each cell type; some may require longer incubations or higher antibody concentrations.

• Mitochondrial Content Normalization: When comparing TMEM11 levels between cell types, normalize to mitochondrial mass using mitochondria-specific markers.

What experimental approaches can reveal the functional significance of TMEM11's interactions with BNIP3 and BNIP3L?

To investigate the functional significance of TMEM11's interactions with BNIP3 and BNIP3L, researchers should consider these experimental approaches:

• Domain Mapping: Generate truncation or point mutants of TMEM11 to identify specific domains required for BNIP3/BNIP3L binding. Test these constructs using co-immunoprecipitation to map interaction interfaces .

• Functional Rescue Experiments: In TMEM11-depleted cells showing mitochondrial morphology defects, express wild-type TMEM11 or mutants deficient in BNIP3/BNIP3L binding to determine if these interactions are necessary for TMEM11's role in mitochondrial morphogenesis .

• Mitophagy Assessment: Since BNIP3 and BNIP3L are known mitophagy receptors, measure mitophagy levels using reporters like mtKeima in cells with normal, depleted, or mutant TMEM11 to determine if TMEM11 regulates mitophagy through these interactions .

• Stress Response Studies: Expose cells to mitochondrial stressors (e.g., CCCP, hypoxia) that typically activate BNIP3/BNIP3L and assess whether TMEM11 is required for their stress-induced functions.

• Proximity-Based Labeling: Implement APEX2-based proximity labeling to identify proteins that associate with TMEM11-BNIP3/BNIP3L complexes under different conditions .

• Live-Cell Imaging: Perform time-lapse microscopy with fluorescently tagged TMEM11, BNIP3, and BNIP3L to visualize dynamic interactions during mitochondrial morphology changes or mitophagy.

• Metabolic Analysis: Compare metabolic profiles of wild-type, TMEM11-depleted, and BNIP3/BNIP3L-depleted cells to identify shared metabolic pathways affected by these proteins .

How can researchers effectively use TMEM11 antibodies in super-resolution microscopy studies?

For effective use of TMEM11 antibodies in super-resolution microscopy, researchers should implement these specialized approaches:

• Fixation Optimization: Test multiple fixation protocols (4% PFA, methanol, or glutaraldehyde-based) to identify which best preserves mitochondrial structure while maintaining TMEM11 antigenicity.

• Antibody Validation: Verify antibody specificity using TMEM11 knockout cells as negative controls and TMEM11-GFP expressing cells as positive controls before proceeding with complex super-resolution experiments .

• Multi-Color Imaging Strategy: Design co-localization experiments with markers for different mitochondrial compartments (TOMM20 for OMM, MIC60 for cristae junctions, HSP60 for matrix) to precisely map TMEM11 distribution .

• Sample Preparation Considerations:

  • For STED microscopy: Use secondary antibodies with specialized dyes (STAR635P, ATTO647N)

  • For STORM/PALM: Consider direct-labeled primary antibodies to increase localization precision

  • For SIM: Ensure high signal-to-noise ratio through optimized blocking and washing steps

• Resolution Calibration: Include fiducial markers of known size to calibrate resolution measurements.

• Quantitative Analysis: Implement specialized software (ImageJ with appropriate plugins, Imaris, or custom scripts) to quantify co-localization, distribution patterns, or clustering behavior of TMEM11 relative to other mitochondrial proteins .

• Correlative Light and Electron Microscopy (CLEM): Consider CLEM approaches combining super-resolution fluorescence with electron microscopy, particularly using APEX2-GFP-TMEM11 constructs that enable both fluorescence detection and electron-dense DAB reaction products .

How might TMEM11 antibodies be utilized in studies of mitochondrial dynamics during cellular stress?

TMEM11 antibodies can be powerful tools for investigating mitochondrial dynamics during cellular stress through these methodological approaches:

• Stress-Induced Redistribution: Monitor TMEM11 localization changes during mitochondrial stress (hypoxia, nutrient deprivation, oxidative stress) using immunofluorescence with super-resolution imaging .

• Post-Translational Modifications: Investigate potential stress-induced modifications of TMEM11 using immunoprecipitation followed by mass spectrometry or Western blotting with modification-specific antibodies.

• Stress-Dependent Interactions: Compare TMEM11 interaction partners under normal versus stress conditions through co-immunoprecipitation and mass spectrometry to identify stress-specific protein complexes .

• Mitochondrial Fragmentation Response: Quantify the correlation between TMEM11 levels/localization and mitochondrial fragmentation during stress using live-cell imaging with TMEM11-GFP and mitochondrial markers .

• Stress Recovery Dynamics: Track TMEM11 during stress recovery phases to determine its role in mitochondrial network restoration using time-lapse microscopy.

• Proteasomal and Mitophagic Degradation: Examine whether TMEM11 is degraded during prolonged stress by treating cells with proteasome inhibitors or mitophagy inducers and quantifying TMEM11 levels.

• Cell-Type Specific Responses: Compare TMEM11 behavior during stress across different cell types to identify tissue-specific roles in stress adaptation.

What methodological approaches can help determine TMEM11's precise topology in the outer mitochondrial membrane?

Determining TMEM11's precise membrane topology requires specialized biochemical and imaging approaches:

• Protease Protection Assays: Treat isolated mitochondria with proteases (trypsin, proteinase K) before and after membrane permeabilization to determine which TMEM11 domains are accessible from different compartments.

• Domain-Specific Antibodies: Generate or acquire antibodies targeting different domains of TMEM11, then perform immunofluorescence or electron microscopy on intact and permeabilized mitochondria to determine domain accessibility.

• Reporter Fusion Constructs: Create fusion constructs with reporters (GFP, APEX2) at different positions within TMEM11, then analyze their localization and accessibility .

• Glycosylation Mapping: Insert glycosylation sites at various positions in TMEM11 and assess which sites become glycosylated (indicating localization in lumen/intermembrane space) versus remaining unmodified (indicating cytosolic/matrix localization).

• Cysteine Accessibility: Implement substituted-cysteine accessibility method (SCAM) by introducing cysteines at different positions and testing their reactivity with membrane-permeable and impermeable sulfhydryl reagents.

• Alkaline Carbonate Extraction: Treat mitochondrial fractions with sodium carbonate at increasing pH (11-12.5) to distinguish peripheral from integral membrane proteins and determine the strength of membrane association .

• Computational Prediction Validation: Validate topology predictions from algorithms like TMHMM and Phobius through experimental approaches listed above.

How can TMEM11 antibodies be integrated into high-throughput screening approaches for mitochondrial function?

Integrating TMEM11 antibodies into high-throughput screening platforms opens new research possibilities:

• Automated Immunofluorescence: Develop automated immunostaining protocols for TMEM11 that can be implemented in high-content imaging systems to screen compounds affecting mitochondrial morphology .

• Multi-Parameter Phenotypic Screening: Combine TMEM11 immunostaining with other mitochondrial markers (membrane potential, ROS production, mitophagy) in multiplexed assays to create comprehensive mitochondrial health profiles.

• Flow Cytometry Applications: Develop cell permeabilization and staining protocols for intracellular TMEM11 detection by flow cytometry, enabling rapid analysis of thousands of cells under different treatment conditions.

• ELISA-Based Compound Screening: Create ELISA-based assays to screen for compounds that affect TMEM11 protein levels or post-translational modifications in cell lysates .

• Protein-Protein Interaction Screens: Implement AlphaScreen or FRET-based assays using labeled TMEM11 antibodies to screen for compounds disrupting or enhancing interactions with partners like BNIP3/BNIP3L .

• CRISPR Screen Validation: Use TMEM11 antibodies to validate hits from genome-wide CRISPR screens targeting mitochondrial morphology or function, particularly for screens identifying TMEM11 as a hit .

• Microfluidic Applications: Incorporate TMEM11 antibody-based detection into microfluidic devices for real-time monitoring of mitochondrial parameters in response to rapidly changing conditions.

What considerations are important when using TMEM11 antibodies in tissue samples for studying mitochondrial pathologies?

When applying TMEM11 antibodies to tissue samples for mitochondrial pathology investigations, researchers should consider:

• Tissue Preservation Methods: Compare different fixation protocols (fresh-frozen, formalin-fixed paraffin-embedded, optimum cutting temperature compound) to determine optimal TMEM11 epitope preservation in tissues.

• Antigen Retrieval Optimization: For FFPE samples, test various antigen retrieval methods (heat-induced epitope retrieval with citrate or EDTA buffers at different pH values) to maximize TMEM11 detection.

• Dual Immunolabeling Strategy: Implement dual labeling with cell-type-specific markers to identify TMEM11 expression patterns across different cell populations within heterogeneous tissues.

• Tissue-Specific Controls: Include tissue from genetic models with TMEM11 deletion or overexpression as controls, particularly when studying pathological samples .

• Quantification Methods: Develop standardized quantification approaches for TMEM11 immunostaining intensity that account for tissue autofluorescence and background.

• Co-localization Analysis: Perform co-localization studies with mitochondrial markers and potential interaction partners relevant to specific pathologies .

• Comparison Across Disease States: Implement case-control designs with matched samples to identify disease-specific alterations in TMEM11 expression or localization.

• Complementary Approaches: Validate immunohistochemistry findings with orthogonal methods such as laser capture microdissection followed by Western blot or quantitative PCR for TMEM11.