TMEM19 Antibody

How does TMEM19 differ from TMEM199?

Despite the similar nomenclature, TMEM19 and TMEM199 are distinct proteins with different structures and functions:

TMEM199 has been shown to have nuclear localization and functions in regulating immune checkpoint proteins such as PD-L1 (CD274), whereas TMEM19's functions remain less extensively characterized in the current literature . When designing experiments targeting either protein, researchers must be careful to select antibodies with validated specificity for the intended target.

Which species demonstrate conserved TMEM19 orthologs?

TMEM19 demonstrates evolutionary conservation across multiple species, making it amenable to comparative studies. Documented orthologs have been identified in:

• Mouse (Mus musculus)

• Rat (Rattus norvegicus)

• Bovine species

• Frog (Xenopus species)

• Zebrafish (Danio rerio)

• Chimpanzee (Pan troglodytes)

• Chicken (Gallus gallus)

This conservation across vertebrate species suggests TMEM19 likely serves a fundamental biological function that has been maintained throughout evolution . When designing cross-species studies, researchers should verify sequence homology to ensure antibody compatibility with the target species.

What are the validated applications for TMEM19 antibodies in research?

TMEM19 antibodies have been validated for several standard immunological detection methods:

When selecting an antibody for a specific application, researchers should prioritize those with validation data for their particular experimental system and application . Published literature citing the antibody for similar applications provides additional validation support.

How should researchers design proper controls when using TMEM19 antibodies?

Proper experimental controls are essential for meaningful interpretation of TMEM19 antibody data:

• Positive controls: Include tissues or cell lines with documented TMEM19 expression.

• Negative controls:

  • Tissue or cells with TMEM19 knocked down or knocked out using siRNA or CRISPR

  • Secondary antibody-only controls to assess background staining

  • Isotype controls to evaluate non-specific binding

• Peptide competition assays: Pre-incubating the antibody with a TMEM19-specific peptide should abolish specific binding.

• Cross-reactivity assessment: Test the antibody against related family members to ensure specificity, particularly to distinguish from TMEM199 .

Implementing these controls helps distinguish genuine TMEM19 signal from artifacts or cross-reactivity, particularly important given the similar nomenclature between TMEM19 and TMEM199.

What optimization strategies are recommended for Western blot detection of TMEM19?

Western blot detection of transmembrane proteins like TMEM19 requires specific technical considerations:

• Sample preparation:

  • Use membrane protein extraction buffers containing appropriate detergents (RIPA or NP-40)

  • Avoid boiling samples, which can cause aggregation of transmembrane proteins

  • Consider inclusion of protease inhibitors to prevent degradation

• Gel selection:

  • Use gradient gels (4-12% or 4-20%) for better resolution

  • For transmembrane proteins, SDS-PAGE with Tris-glycine or Tris-tricine systems may provide better separation

• Transfer optimization:

  • Extended transfer times or semi-dry transfer systems may improve efficiency

  • Consider using PVDF membranes which often perform better for hydrophobic proteins

  • Addition of SDS (0.1%) in transfer buffer can improve transfer of transmembrane proteins

• Blocking and antibody incubation:

  • Test both milk and BSA-based blocking solutions (BSA often works better for phospho-specific detection)

  • Extended primary antibody incubation at 4°C may improve signal quality

  • Optimize antibody dilutions based on manufacturer recommendations

What methodological approaches are most effective for studying TMEM19 function?

Given the limited characterized functions of TMEM19, several methodological approaches can be employed:

• Loss-of-function studies:

  • siRNA or shRNA knockdown to assess phenotypic changes

  • CRISPR-Cas9 gene editing for complete knockout

  • Monitor effects on cell viability, morphology, and related pathways

• Gain-of-function studies:

  • Overexpression of tagged TMEM19 constructs

  • Inducible expression systems for temporal control

• Localization studies:

  • Immunofluorescence with co-localization markers for different cellular compartments

  • Live-cell imaging with fluorescently-tagged TMEM19

  • Subcellular fractionation followed by Western blot analysis

• Interaction studies:

  • Co-immunoprecipitation to identify binding partners

  • Proximity labeling approaches (BioID or APEX)

  • Yeast two-hybrid or mammalian two-hybrid screens

Unlike TMEM199, which has been reported to affect immune regulation and PDL1 expression, specific functions of TMEM19 require further investigation using these methodological approaches .

How can researchers address potential cross-reactivity between TMEM19 and TMEM199 antibodies?

Given the similar nomenclature, cross-reactivity is a significant concern:

• Sequence alignment analysis:

  • Compare amino acid sequences of both proteins to identify unique epitopes

  • Select antibodies raised against regions with minimal homology

• Validation experiments:

  • Test antibodies on samples with selective knockdown of each protein

  • Perform peptide competition assays with specific peptides from each protein

  • Compare staining patterns with multiple antibodies targeting different epitopes

• Mass spectrometry validation:

  • Immunoprecipitate the target protein and confirm identity by mass spectrometry

  • This approach definitively identifies the captured protein

• Recombinant protein controls:

  • Test antibody reactivity against purified recombinant TMEM19 and TMEM199

  • Evaluate specificity and potential cross-reactivity in controlled conditions

What is the documented tissue distribution pattern of TMEM19?

TMEM19 demonstrates broad tissue expression patterns, though with varying levels across different tissues:

For accurate assessment of TMEM19 expression in specific experimental contexts, researchers should:

• Utilize tissue-specific databases like Human Protein Atlas or GTEx

• Validate expression through multiple approaches (RT-qPCR, Western blot, IHC)

• Consider both mRNA and protein detection methods, as post-transcriptional regulation may affect correlation between transcript and protein levels

What are the best practices for quantifying TMEM19 expression changes in experimental settings?

For accurate quantification of TMEM19 expression:

• RNA-level analysis:

  • RT-qPCR with validated primers spanning exon junctions

  • Consider multiple reference genes for normalization

  • RNA-seq for comprehensive transcriptomic analysis

• Protein-level analysis:

  • Western blot with proper loading controls (β-actin, GAPDH, or preferably other membrane proteins)

  • Quantitative flow cytometry if cell surface expression is confirmed

  • ELISA for high-throughput quantitative analysis

• Statistical considerations:

  • Perform biological replicates (minimum n=3)

  • Apply appropriate statistical tests based on data distribution

  • Consider power analysis to determine sufficient sample size

• Reporting standards:

  • Follow MIQE guidelines for qPCR experiments

  • Include complete methodology and antibody validation information

  • Report both statistical and biological significance of findings

How might differential isoform expression of TMEM19 impact experimental design and interpretation?

With up to two reported isoforms of TMEM19, researchers should consider:

• Isoform-specific detection:

  • Design primers or select antibodies that can distinguish between isoforms

  • Consider isoform-specific functions that may be masked in bulk analyses

• Experimental approaches:

  • RNA-seq with sufficient depth for isoform quantification

  • Targeted proteomics to detect isoform-specific peptides

  • Cloning and expression of individual isoforms to assess functional differences

• Data interpretation considerations:

  • Different isoforms may localize to different subcellular compartments

  • Isoforms may interact with different binding partners

  • Tissue-specific or condition-specific isoform expression patterns may exist

What methodological approaches should be used to investigate potential roles of TMEM19 in disease models?

To investigate TMEM19's potential roles in disease:

• Expression analysis in disease tissues:

  • Compare TMEM19 levels in normal versus disease tissues

  • Analyze correlation with disease progression or clinical outcomes

  • Consider single-cell approaches to address cellular heterogeneity

• Functional models:

  • Generate knockout or knockdown models in relevant cell types

  • Assess phenotypic consequences on disease-relevant pathways

  • Complement in vitro findings with appropriate animal models

• Translational approaches:

  • Examine potential as biomarker through retrospective cohort studies

  • Evaluate correlation with established disease markers

  • Consider therapeutic targeting potential if disease association is established

Unlike TMEM199, which has documented roles in immune regulation and cancer, specific disease associations for TMEM19 require further investigation using these methodological approaches .