TMEM154 Antibody

What is TMEM154 and what is its relevance in research?

TMEM154 is a 128 amino acid transmembrane protein encoded by a gene located on human chromosome 4. This protein plays a crucial role in immune response regulation, particularly in the context of viral infections and T cell response modulation . In veterinary research, TMEM154 has gained significance following the discovery of its variants affecting susceptibility to small ruminant lentiviruses (SRLVs) in sheep, causing diseases such as maedi-visna and ovine progressive pneumonia . The protein's involvement in immune signaling pathways makes it a valuable research target for understanding disease mechanisms and developing potential therapeutic strategies for conditions requiring immune modulation .

What applications are TMEM154 antibodies suitable for?

TMEM154 antibodies are versatile research tools suitable for multiple experimental applications. Commercial antibodies such as the mouse monoclonal TMEM154 Antibody (F-8) can be used for western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . Polyclonal antibodies like 24812-1-AP are specifically optimized for immunohistochemistry (IHC) and ELISA applications . When selecting an antibody for your research, consider the specific application requirements and sample types. For IHC applications, recommended dilutions typically range from 1:20 to 1:200, though this should be optimized for your specific experimental system .

How should TMEM154 antibodies be stored and handled for optimal results?

For maximum stability and activity retention, TMEM154 antibodies should be stored at -20°C. Most commercial preparations remain stable for one year after shipment when stored properly . Many TMEM154 antibodies are supplied in storage buffers containing preservatives such as sodium azide (0.02%) and stabilizers like glycerol (50%) at pH 7.3 . For smaller volume products (e.g., 20μl sizes), manufacturers often include 0.1% BSA as an additional stabilizer . When working with these antibodies, aliquoting is generally unnecessary for -20°C storage, reducing the risk of contamination from repeated freeze-thaw cycles. Always follow manufacturer-specific recommendations, as formulations may vary between suppliers.

What methodological considerations are important when using TMEM154 antibodies for IHC?

When performing immunohistochemistry with TMEM154 antibodies, antigen retrieval is a critical step that significantly impacts staining quality. For optimal results with polyclonal antibodies like 24812-1-AP, antigen retrieval with TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 may serve as an alternative . The table below summarizes key methodological parameters for IHC detection of TMEM154:

Researchers should always perform preliminary titration experiments to determine the optimal antibody concentration for their specific tissue samples, as expression levels can vary significantly between tissue types .

How can researchers validate the specificity of TMEM154 antibodies?

Validating antibody specificity is essential for generating reliable research data. For TMEM154 antibodies, multiple validation approaches should be employed:

• Positive and negative controls: Use tissues with known TMEM154 expression profiles, such as human kidney tissue (positive) for IHC applications .

• Blocking peptide experiments: Commercial neutralizing peptides (such as sc-398803 P) can be used to confirm binding specificity .

• Genetic validation: In studies where possible, compare antibody reactivity in wild-type versus TMEM154 knockout models or cells.

• Multiple detection methods: Confirm expression using orthogonal techniques (e.g., validate IHC findings with western blotting).

• Isotype controls: Include appropriate isotype controls (e.g., rabbit IgG for polyclonal rabbit antibodies) to assess non-specific binding.

Each validation approach strengthens confidence in antibody specificity, and researchers should document these validation steps in their methods sections.

How can TMEM154 antibodies be used to study the relationship between TMEM154 variants and viral susceptibility?

TMEM154 genetic variants have been associated with susceptibility to viral infections, particularly in sheep. Researchers can use TMEM154 antibodies alongside genotyping to investigate protein expression differences between variants and correlate these with infection outcomes .

A comprehensive approach would involve:

• Genotyping subjects for known TMEM154 polymorphisms (such as E35K, R4A∆, T44M, or N70I in sheep)

• Quantifying protein expression using calibrated immunoassays with TMEM154 antibodies

• Assessing viral loads using PCR-based methods

• Performing immunolocalization studies to determine if variant proteins show different cellular distribution patterns

This integrated approach has successfully demonstrated that sheep with the K35 variant of TMEM154 show increased resistance to SRLV infection compared to those with the E35 variant . Similar methodologies could be applied to study other TMEM154 variants and their relationship to disease susceptibility in different species.

What approaches can be used to investigate TMEM154's role in immune signaling pathways?

TMEM154 has been implicated in the regulation of immune signaling pathways, particularly in viral infection contexts . To investigate its mechanistic role, researchers can employ TMEM154 antibodies in the following experimental approaches:

• Co-immunoprecipitation studies: Using TMEM154 antibodies to pull down the protein and identify interaction partners through mass spectrometry.

• Signaling pathway analysis: Combining TMEM154 knockdown/overexpression with phospho-specific antibodies to map affected pathways.

• Live cell imaging: Employing fluorescently tagged TMEM154 antibodies to track protein localization during immune activation.

• Flow cytometry: Analyzing TMEM154 expression changes in specific immune cell populations during immune responses.

• ChIP-seq analysis: Investigating transcriptional regulation of TMEM154 in different immune contexts.

These approaches can help elucidate how TMEM154 contributes to immune response regulation and potentially identify intervention points for therapeutic development .

How can researchers effectively detect and characterize TMEM154 SNPs in population studies?

The identification of single nucleotide polymorphisms (SNPs) in TMEM154 requires a systematic approach, particularly when studying population-level associations with disease susceptibility. Based on current research methodologies:

• DNA extraction and quality control: Extract high-quality DNA from blood or tissue samples, ensuring sufficient purity (260/280 ratio of 1.8-2.0).

• PCR amplification: Design primers flanking regions of interest in the TMEM154 gene, including exons 1 and 2 where many functional SNPs are located .

• Sanger sequencing: Sequence PCR products to identify SNPs. In the Polish sheep study, this approach identified 10 mutations within the TMEM154 locus .

• SNP validation: Compare identified SNPs with annotated variants in databases such as EVA (EMBL-EBI) .

• Statistical analysis: Calculate allele frequencies and test for Hardy-Weinberg equilibrium across studied populations.

This approach has been successfully employed to identify novel TMEM154 polymorphisms and characterize known variants in sheep populations, as demonstrated in the first report of SNPs in TMEM154 in Polish sheep flocks .

What methods are most effective for correlating TMEM154 genotypes with viral resistance phenotypes?

To establish robust correlations between TMEM154 genotypes and viral resistance, researchers should implement a comprehensive phenotyping and genotyping strategy:

• Infection status determination: Use both serological (antibody-based) and molecular (PCR-based) methods to confirm infection status, as demonstrated in the study of SRLV in Polish sheep .

• Proviral load quantification: Employ quantitative PCR to measure viral burden in infected individuals, allowing for assessment of resistance levels rather than binary infected/uninfected categories .

• Genotype-phenotype statistical analyses: Calculate relative risk scores to quantify the association between specific TMEM154 genotypes and infection outcomes. Consider breed-specific analyses as genetic backgrounds may influence associations .

• Multivariate analyses: Control for confounding factors such as age, sex, and environmental exposure.

This methodological approach has successfully identified significant associations between specific TMEM154 genotypes (e.g., E35K polymorphism) and SRLV susceptibility, with AA homozygotes (encoding KK isoform) showing increased resistance to infection .

How can researchers address cross-reactivity concerns when using TMEM154 antibodies across species?

Cross-species reactivity remains a significant challenge when using TMEM154 antibodies. While some commercial antibodies claim reactivity with mouse, rat, and human TMEM154 , researchers should employ the following validation strategies when using these antibodies in different species:

• Sequence homology analysis: Compare TMEM154 protein sequences across target species to predict potential cross-reactivity.

• Epitope mapping: Identify the specific epitope recognized by the antibody and assess its conservation across species.

• Validation experiments: Perform western blots using recombinant TMEM154 proteins from each species of interest.

• Positive controls: Include known positive samples from each species in initial experiments.

• Optimization of conditions: Adjust antibody concentration, incubation times, and buffer conditions for each species.

Remember that even when an antibody is reported to cross-react with multiple species, sensitivity may vary significantly, requiring species-specific protocol optimization.

What are the common challenges in detecting low-abundance TMEM154 in tissue samples?

Detecting low-abundance proteins like TMEM154 in tissue samples presents several technical challenges. Researchers can implement these strategies to enhance detection:

• Signal amplification systems: Use polymer-based detection systems or tyramide signal amplification to enhance sensitivity in IHC applications.

• Optimized antigen retrieval: For IHC, thoroughly optimize antigen retrieval conditions, as recommended protocols suggest using TE buffer at pH 9.0 for TMEM154 detection .

• Concentration of samples: For western blotting, concentrate protein samples using immunoprecipitation before analysis.

• Reduced background strategies: Implement thorough blocking protocols and include appropriate controls to distinguish specific signal from background.

• Alternative detection methods: Consider more sensitive techniques like proximity ligation assay (PLA) or branched DNA signal amplification.

When working with challenging samples, it may be necessary to test multiple antibody clones, as different epitopes may be more accessible depending on tissue preparation methods.

How might TMEM154 research contribute to genetic selection programs for disease resistance in livestock?

The identification of TMEM154 variants associated with resistance to small ruminant lentiviruses offers promising applications for selective breeding programs in sheep:

• Marker-assisted selection: Using genetic markers like the E35K polymorphism to select breeding stock with increased resistance to SRLV infection .

• Genomic prediction models: Incorporating TMEM154 genotype data into broader genomic selection models that account for multiple resistance loci.

• Economic impact assessment: Evaluating the cost-benefit ratio of implementing TMEM154-based selection in commercial flocks.

• Integrated control strategies: Combining genetic selection with management practices to control SRLV spread.

Recent research in Polish sheep flocks demonstrated that selecting SRLV-resistant animals based on TMEM154 polymorphism analysis is promising, though validation in larger populations is needed . This approach could potentially reduce the prevalence of SRLV infections in sheep populations, improving animal welfare and reducing economic losses in the industry.

What is the potential role of TMEM154 in human immunology and disease?

While much of the published research has focused on TMEM154's role in veterinary contexts, emerging evidence suggests potential significance in human immunology:

• T cell response modulation: TMEM154 has been implicated in modulating T cell responses, suggesting potential relevance in autoimmune diseases and cancer immunotherapy .

• Viral susceptibility: Given its role in SRLV susceptibility in sheep, human TMEM154 variants may influence susceptibility to human lentiviruses or other viral pathogens.

• Inflammatory signaling: As a transmembrane protein involved in immune signaling pathways, TMEM154 may participate in inflammatory cascades relevant to various human diseases.

• Therapeutic target potential: Understanding TMEM154's role in immune regulation could lead to novel therapeutic approaches for diseases requiring immune modulation .

Future research directions may include investigating associations between human TMEM154 polymorphisms and disease susceptibility, exploring TMEM154 as a biomarker for immune-related conditions, and developing therapeutic strategies targeting TMEM154-dependent pathways.