TMEM91 Antibody

What is TMEM91 and why is it relevant to current research?

TMEM91 (Transmembrane protein 91) is a human protein also known as Dispanin subfamily C member 3 (DSPC3). While its complete biological function remains under investigation, recent studies have identified TMEM91 as one of six genes associated with COVID-19 severity through RNA sequencing analysis of respiratory tract samples . This association was identified through statistical tests that compared severe COVID-19 cases (patients requiring mechanical ventilation or extracorporeal membrane oxygenation) with non-severe cases. TMEM91 was found alongside other genes including RPL15, BACE1-AS, CEPT1, EIF4G1, and TBCK, suggesting potential roles in disease mechanisms that warrant further investigation .

What validated applications are supported by commercial TMEM91 antibodies?

Commercial TMEM91 antibodies have been validated for several standard laboratory techniques:

These applications have been validated with specific human cell lines and tissues, with optimal dilutions varying by application and specific antibody product .

How should I design proper controls when using TMEM91 antibodies in Western blotting?

When designing Western blot experiments with TMEM91 antibodies, implement both positive and negative controls to ensure reliable interpretation:

For positive controls:

• Use lysates from cells known to express TMEM91, such as A549 human lung carcinoma cells, which have been validated with commercial antibodies .

• The predicted band size for human TMEM91 is approximately 18 kDa , which should be your primary target band.

For negative controls:

• Include samples where TMEM91 is knocked down using siRNA or CRISPR.

• Use cell lines that do not express TMEM91 (though specific negative cell lines are not explicitly mentioned in the provided data).

• Perform peptide competition assays by pre-incubating the antibody with the immunogen peptide.

Additionally, always include loading controls (such as GAPDH or β-actin) to normalize protein loading across lanes, and consider running a molecular weight marker to accurately identify your target band. When interpreting results, be aware that post-translational modifications might cause slight deviations from the predicted molecular weight.

What are the critical factors for successful immunohistochemistry (IHC) using TMEM91 antibodies?

For optimal IHC results with TMEM91 antibodies, consider these methodological factors:

• Tissue preparation: Use properly fixed (typically paraffin-embedded) human tissue samples. Successful staining has been reported with human liver cancer and small intestine tissues .

• Antigen retrieval: This critical step unmasks epitopes potentially hidden during fixation. Since TMEM91 is a transmembrane protein, heat-induced epitope retrieval in citrate buffer (pH 6.0) is often effective.

• Antibody dilution: Start with the manufacturer's recommended range (typically 1/20-1/200 for IHC) , and optimize for your specific tissue samples. For the antibody ab236864, a 1/100 dilution has been successfully used for human tissues .

• Detection system: Use an appropriate secondary antibody system compatible with your primary antibody (typically anti-rabbit for TMEM91 antibodies).

• Controls: Include positive control tissues (liver or small intestine based on validated results), negative controls (omission of primary antibody), and if possible, TMEM91-depleted tissues as biological negative controls.

• Counterstaining: Use appropriate nuclear counterstaining (typically hematoxylin) to provide cellular context for the TMEM91 signal.

Proper optimization of these factors will help ensure specific and reproducible TMEM91 staining in your tissue samples.

How can I validate the specificity of TMEM91 antibody staining in immunofluorescence experiments?

Validating specificity in immunofluorescence requires multiple complementary approaches:

• Genetic knockdown/knockout validation: The gold standard for antibody validation involves comparing staining between wild-type cells and cells where TMEM91 has been knocked down (siRNA) or knocked out (CRISPR/Cas9). A specific antibody will show significantly reduced signal in the knockdown/knockout samples.

• Overexpression controls: Transfect cells with a TMEM91 expression vector (ideally with an orthogonal tag like GFP or FLAG) and confirm co-localization of the antibody signal with the tagged protein.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide (recombinant TMEM91 fragments aa 1-97) before staining. Specific binding should be blocked by this competition.

• Subcellular localization consistency: As a transmembrane protein, TMEM91 should exhibit a pattern consistent with membrane localization. In MCF7 cells, for example, TMEM91 antibodies have demonstrated specific staining patterns .

• Multi-antibody concordance: If possible, compare staining patterns using multiple antibodies targeting different epitopes of TMEM91.

• Secondary antibody controls: Always include a control with only the secondary antibody to identify potential non-specific background.

For TMEM91 immunofluorescence, successful staining has been demonstrated in MCF7 cells using a 1/100 dilution of specific antibodies followed by Alexa Fluor 488-conjugated secondary antibodies .

What are the recommended approaches for multiplexing TMEM91 detection with other markers?

Multiplexing TMEM91 with other proteins requires careful planning:

• Primary antibody compatibility: When selecting antibodies for co-detection with TMEM91, choose primary antibodies from different host species than your TMEM91 antibody (which is typically rabbit-derived). For example, pair rabbit anti-TMEM91 with mouse, goat, or rat antibodies against your other proteins of interest.

• Sequential immunostaining: For challenging multiplex staining, consider sequential staining protocols:

  • Apply and detect the first primary antibody

  • Elute or quench the first set of antibodies

  • Apply and detect the second primary antibody

  • This approach minimizes cross-reactivity issues

• Spectral separation: Choose fluorophores with minimal spectral overlap:

  • For rabbit anti-TMEM91, Alexa Fluor 488 (green) has been successfully used

  • Pair with red (Alexa 594/647) or far-red fluorophores for other targets

  • Include single-color controls to confirm specificity

• Organelle markers: Since TMEM91 is a transmembrane protein, consider co-staining with established membrane compartment markers to determine its precise subcellular localization.

• Cross-validation: Confirm co-localization or expression pattern relationships through complementary techniques such as proximity ligation assays or co-immunoprecipitation where appropriate.

When implementing multiplexed detection, always include appropriate compensation controls if using flow cytometry or spectral imaging systems to account for potential bleed-through between channels.

How should I interpret multiple bands in Western blots using TMEM91 antibodies?

When encountering multiple bands in TMEM91 Western blots, consider these interpretation guidelines:

• Expected band: The predicted molecular weight of human TMEM91 is approximately 18 kDa . This should be your primary target band.

• Potential causes of multiple bands:

  • Post-translational modifications: Phosphorylation, glycosylation, or other modifications can alter protein migration

  • Isoforms: Alternative splicing may generate multiple TMEM91 variants

  • Protein degradation: Partial proteolysis can produce fragments

  • Oligomerization: Protein complexes that resist denaturation

  • Non-specific binding: Cross-reactivity with other proteins

• Validation approaches:

  • Lysate treatment: Use phosphatases or glycosidases to determine if modifications cause band shifts

  • Loading controls: Ensure equal loading and transfer efficiency across samples

  • Peptide competition: Pre-incubate antibody with immunizing peptide; specific bands should disappear

  • TMEM91 knockdown: Specific bands should decrease in intensity in knockdown samples

• Documentation best practices: Always document all observed bands, not just the expected ones, and include molecular weight markers in your images. Compare your results with previous literature and antibody documentation to identify consistencies and discrepancies.

The specificity of commercial TMEM91 antibodies has been validated with human cell lysates (e.g., A549 cells), showing the expected band at approximately 18 kDa , which should serve as your primary reference point.

What are common pitfalls in TMEM91 immunostaining and how can they be resolved?

Common challenges in TMEM91 immunostaining and their solutions include:

For TMEM91 specifically, successful immunostaining has been reported in human liver cancer tissue, small intestine tissue, and MCF7 cells using a 1/100 dilution . When optimizing, begin with these validated conditions and adjust systematically while maintaining appropriate controls.

How is TMEM91 being investigated in COVID-19 research, and what methodological approaches are recommended?

Recent research has identified TMEM91 as one of six genes associated with COVID-19 severity , suggesting potential roles in disease mechanisms. For researchers investigating this connection:

• Study design approaches:

  • Compare gene expression between severe and non-severe COVID-19 patients using RNA sequencing of respiratory tract samples

  • Categorize severity based on clinical parameters (e.g., need for mechanical ventilation or extracorporeal membrane oxygenation)

  • Control for confounding factors including age, sex, and comorbidities

• Methodological recommendations:

  • Analyze TMEM91 expression alongside other identified genes (RPL15, BACE1-AS, CEPT1, EIF4G1, and TBCK)

  • Employ both logistic regression and Kolmogorov-Smirnov statistical tests for robust analysis

  • Validate RNA-seq findings with RT-PCR or protein-level analysis using TMEM91 antibodies in patient samples

• Functional investigation approaches:

  • Examine TMEM91 protein expression in respiratory epithelia using validated antibodies via IHC/IF

  • Investigate potential interactions between TMEM91 and viral proteins through co-immunoprecipitation

  • Consider TMEM91 knockdown/overexpression studies in relevant cell models to assess functional impact on viral replication or inflammatory responses

• Integration with other findings:

  • Gene ontology analysis of differentially expressed genes in COVID-19 has shown involvement in nervous system diseases , suggesting broader systemic effects

  • Consider investigating TMEM91 in neural cell models alongside respiratory epithelial models

This emerging research area highlights the importance of correlating gene expression data with protein-level analysis using validated TMEM91 antibodies in relevant tissue and cell types.

What experimental design considerations are important when studying TMEM91 in different disease contexts?

When investigating TMEM91 across disease contexts, consider these experimental design principles:

• Cohort selection and characterization:

  • Define clear inclusion/exclusion criteria and disease classifications

  • Document demographic information (age, sex, ethnicity) as these may influence gene expression

  • For COVID-19 studies, stratify by clinical parameters like ventilation requirements

  • For potential diabetes connections, consider factors like age at first antibody appearance and genetic risk

• Sample collection and processing standardization:

  • Use consistent collection protocols to minimize technical variability

  • Document the interval between clinical events and sample collection

  • Process all samples using identical workflows to ensure comparability

• Multi-omics integration strategy:

  • Correlate TMEM91 transcript levels (RNA-seq/qPCR) with protein expression (using validated antibodies)

  • Consider examination in multiple tissue types depending on disease context

  • For respiratory diseases, include both upper and lower respiratory tract samples

  • For metabolic conditions, consider relevant metabolic tissues

• Functional validation approaches:

  • Develop disease-relevant cell models expressing or lacking TMEM91

  • Assess physiological responses under disease-mimicking conditions

  • Consider animal models where appropriate to validate human findings

• Statistical considerations:

  • Calculate appropriate sample sizes based on expected effect sizes

  • Account for multiple testing when profiling TMEM91 alongside other markers

  • Control for relevant covariates in statistical models

  • Consider longitudinal sampling where feasible to capture disease progression

By implementing these rigorous experimental design principles, researchers can generate more reliable and translatable findings regarding TMEM91's role across different disease contexts.

How can I design experiments to investigate potential TMEM91 protein-protein interactions?

To investigate TMEM91 protein-protein interactions, implement these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use validated TMEM91 antibodies (such as those from Abcam, Abbexa, or Biomatik) for pull-down experiments

  • Optimize lysis conditions to preserve membrane protein interactions (consider mild detergents like CHAPS or digitonin)

  • Perform reciprocal Co-IPs with antibodies against suspected interaction partners

  • Include appropriate negative controls (IgG, irrelevant antibodies)

  • Validate findings with western blot analysis using specific antibodies against both TMEM91 and interaction partners

• Proximity-based methods:

  • Proximity Ligation Assay (PLA): Detect in situ protein interactions within 40nm distance

  • BioID or APEX2: Employ proximity-dependent biotinylation to identify proteins in close proximity to TMEM91

  • FRET/BRET: For direct interaction studies using fluorescent/bioluminescent fusion proteins

• Protein complementation assays:

  • Split-YFP, split-luciferase, or split-ubiquitin systems for membrane protein interactions

  • Express TMEM91 fused to one fragment and potential partners fused to complementary fragments

• Mass spectrometry-based approaches:

  • Immunoprecipitate TMEM91 from relevant cell types (A549, MCF7)

  • Analyze co-precipitating proteins using high-sensitivity mass spectrometry

  • Apply appropriate filtering against common contaminant databases

• Membrane-specific considerations:

  • As TMEM91 is a transmembrane protein, standard interaction assays may require modifications

  • Consider crosslinking approaches to stabilize transient membrane protein interactions

  • Use appropriate detergents that maintain membrane protein structure while allowing solubilization

When designing these experiments, cell types with validated TMEM91 expression (such as A549 lung carcinoma or MCF7 breast cancer cells) should be prioritized as experimental systems.

What approaches can be used to investigate TMEM91 localization and trafficking in cells?

To study TMEM91 localization and trafficking, employ these methodological strategies:

• High-resolution microscopy approaches:

  • Confocal microscopy: Use validated TMEM91 antibodies (1/50-1/200 dilution) with appropriate membrane compartment markers

  • Super-resolution techniques (STORM, PALM, STED): Achieve nanoscale resolution of TMEM91 distribution

  • Live-cell imaging: Create fluorescent protein fusions (e.g., TMEM91-GFP) for real-time trafficking studies

• Subcellular fractionation and biochemical analysis:

  • Separate cellular compartments (plasma membrane, endosomes, Golgi, etc.)

  • Analyze TMEM91 distribution across fractions via Western blotting

  • Include compartment-specific markers as controls (e.g., Na⁺/K⁺-ATPase for plasma membrane)

• Co-localization studies:

  • Pair TMEM91 antibodies with established markers for:

    • Plasma membrane (e.g., WGA, Na⁺/K⁺-ATPase)

    • Endosomes (e.g., Rab5, Rab7, Rab11)

    • Golgi apparatus (e.g., GM130, TGN46)

    • ER (e.g., calnexin, PDI)

  • Quantify co-localization using appropriate metrics (Pearson's correlation, Manders' overlap)

• Trafficking perturbation approaches:

  • Use temperature blocks (e.g., 15°C, 20°C) to arrest trafficking at specific compartments

  • Apply chemical inhibitors of trafficking pathways (e.g., Brefeldin A, monensin)

  • Employ dominant-negative Rab/Arf GTPases to disrupt specific trafficking steps

  • Monitor TMEM91 redistribution following these perturbations

• Endocytosis and recycling assays:

  • Surface biotinylation to track internalization rates

  • Antibody uptake assays if TMEM91 has an extracellular epitope

  • FRAP (Fluorescence Recovery After Photobleaching) for lateral mobility studies

When implementing these approaches, MCF7 cells have been validated for TMEM91 immunofluorescence studies and would serve as an appropriate initial model system for localization studies.

What new methodologies are being developed for studying TMEM91 function?

While the search results don't specifically mention novel methodologies for TMEM91, researchers can apply cutting-edge approaches based on current understanding of membrane protein analysis:

• CRISPR-based functional genomics:

  • CRISPR knockout/knockin strategies for precise TMEM91 manipulation

  • CRISPRi/CRISPRa for reversible expression modulation

  • CRISPR base or prime editing for introducing specific TMEM91 mutations

  • CRISPR screens to identify synthetic lethal interactions or functional partners

• Advanced proteomics approaches:

  • Thermal proteome profiling to identify TMEM91 interactions and drug targets

  • Crosslinking mass spectrometry (XL-MS) to map structural relationships

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to analyze conformational dynamics

  • Targeted proteomics with parallel reaction monitoring for precise quantification

• Organoid and 3D culture systems:

  • Study TMEM91 in physiologically relevant tissue contexts

  • Patient-derived organoids to examine disease-specific alterations

  • Co-culture systems to investigate intercellular communication roles

• Single-cell technologies:

  • Single-cell RNA-seq to reveal cell-type specific expression patterns

  • Single-cell proteomics to analyze TMEM91 protein levels at cellular resolution

  • Spatial transcriptomics to map TMEM91 expression across tissue architecture

• Computational approaches:

  • AI-based structure prediction (AlphaFold) for TMEM91 structural insights

  • Network analysis to position TMEM91 in broader cellular systems

  • Molecular dynamics simulations to study membrane interactions

These emerging methodologies could provide new insights into TMEM91 function, particularly in the context of its potential role in COVID-19 pathogenesis and other disease states, though validation with established techniques using well-characterized antibodies remains essential.

How can transcriptomic data be integrated with TMEM91 protein analysis for comprehensive understanding?

Integrating transcriptomic data with protein-level analysis of TMEM91 requires systematic methodological approaches:

• Multi-omic correlation strategies:

  • Compare TMEM91 mRNA expression (via RNA-seq or qPCR) with protein levels (via Western blot or immunostaining)

  • Quantify both absolute levels and relative changes across conditions

  • Calculate correlation coefficients to assess transcript-protein relationship

  • Consider time-course experiments to capture potential temporal disconnects

• Single-cell multi-omics:

  • Apply CITE-seq or similar technologies that enable simultaneous measurement of transcripts and proteins

  • Use computational approaches to integrate and visualize multi-modal data

  • Identify cell populations with discordant TMEM91 mRNA and protein patterns

• Functional validation of transcriptomic findings:

  • Validate genes co-expressed with TMEM91 in COVID-19 studies at the protein level

  • Confirm expression patterns of the six-gene signature (RPL15, BACE1-AS, CEPT1, EIF4G1, TMEM91, TBCK) in patient samples

  • Correlate expression with clinical parameters and disease severity markers

• Regulatory network analysis:

  • Identify transcription factors regulating TMEM91 expression

  • Verify regulatory relationships using ChIP-seq or similar techniques

  • Manipulate identified regulators to confirm impact on TMEM91 protein levels

• Translation efficiency assessment:

  • Employ polysome profiling or ribosome profiling to assess TMEM91 translation

  • Compare with total mRNA levels to identify potential translational regulation

  • Investigate RNA-binding proteins that might influence TMEM91 translation

• Data integration frameworks:

  • Apply computational methods like MOFA (Multi-Omics Factor Analysis) or similar tools

  • Generate integrated visualizations that highlight relationships between transcriptomic and proteomic findings

  • Develop predictive models that incorporate both data types

By systematically integrating transcriptomic and protein-level analyses, researchers can build a more comprehensive understanding of TMEM91 biology, particularly in disease contexts like COVID-19 where it has shown differential expression patterns .

What are the best practices for standardizing TMEM91 antibody experiments across different laboratories?

To ensure reproducibility of TMEM91 antibody experiments across laboratories, implement these standardization practices:

• Antibody validation and reporting:

  • Document complete antibody information: manufacturer, catalog number, lot number, clone/polyclonal designation

  • For commercial antibodies, refer to specific validated products (e.g., ab236864, CAC13972)

  • Verify antibody specificity using appropriate controls (knockdown/knockout, peptide competition)

  • Report validation results following standardized guidelines (e.g., RRID identifiers)

• Experimental protocol standardization:

  • For Western blot: Standardize protein extraction methods, loading amounts (typically 20-30 μg), dilutions (1/1000-1/5000) , and exposure times

  • For IHC/ICC: Standardize fixation procedures, antigen retrieval methods, antibody dilutions (1/20-1/200) , and incubation times/temperatures

  • For ELISA: Establish standard curves with recombinant TMEM91 protein

  • Document all protocol details in methods sections using detailed, reproducible language

• Reference samples and controls:

  • Establish common positive control samples (e.g., A549 or MCF7 cell lysates)

  • Implement standardized negative controls (e.g., TMEM91 knockout cells, IgG controls)

  • Consider developing shared reference materials across laboratories

  • Document expected results with these reference samples (e.g., 18 kDa band intensity)

• Image acquisition and analysis standardization:

  • Define standard image acquisition parameters (exposure, gain, resolution)

  • Implement consistent quantification methods (e.g., normalized band intensity, H-score for IHC)

  • Use digital image analysis tools with defined parameters

  • Share unprocessed original images alongside analyzed results

• Reporting standards:

  • Follow field-specific reporting guidelines (e.g., MDAR, ARRIVE)

  • Document all statistical methods and sample sizes

  • Report both positive and negative results

  • Include detailed supplementary methods sections in publications

By implementing these standardization practices, researchers can improve reproducibility of TMEM91 antibody experiments and facilitate meaningful meta-analysis across studies.

How can I validate TMEM91 antibody specificity when working with tissues or cells not previously tested?

When extending TMEM91 antibody use to new tissue or cell types, implement this comprehensive validation workflow:

• Initial bioinformatic assessment:

  • Confirm TMEM91 expression in your target tissue/cell type using transcriptomic databases

  • Check for tissue-specific isoforms or variants that might affect antibody binding

  • Assess homology with related proteins that could cause cross-reactivity

• Gradient of validation approaches:

  • Genetic modification: Generate TMEM91 knockdown/knockout in your target system as negative controls

  • Overexpression: Create TMEM91-overexpressing samples as positive controls

  • Peptide competition: Pre-incubate antibody with the immunizing peptide (aa 1-97 of TMEM91)

  • Orthogonal detection: Compare results using antibodies targeting different TMEM91 epitopes

• Multi-technique concordance:

  • Compare protein detection across multiple methods:

    • Western blot (expected 18 kDa band)

    • Immunostaining patterns

    • Mass spectrometry validation where feasible

  • Consistent results across techniques increase confidence in specificity

• Titration experiments:

  • Perform antibody dilution series to identify optimal concentration

  • For Western blot: Test range from 1/500-1/5000

  • For IHC/IF: Test range from 1/20-1/200

  • Evaluate signal-to-noise ratio at each concentration

  • Specific signals should decrease proportionally with dilution

• Cross-species validation (if applicable):

  • If extending to non-human tissues, align TMEM91 sequences to assess epitope conservation

  • Test against samples with known TMEM91 expression patterns in that species

  • Consider species-specific positive and negative controls

• Documentation standards:

  • Record all validation steps with appropriate images/data

  • Document antibody performance characteristics for your specific tissue/cell type

  • Share validation data to build community knowledge