TMEFF1 Antibody

What is TMEFF1 and what are its primary biological functions?

TMEFF1 (Tomoregulin-1) is a transmembrane protein that functions as a neuron-specific restriction factor against herpes simplex virus 1 (HSV-1) infection in the brain. It acts by blocking viral entry through prevention of the association between viral glycoprotein D (gD) and its cell surface receptor NECTIN1, thereby inhibiting fusion of the virus and cell membrane . Additionally, TMEFF1 prevents the association between viral glycoprotein B (gB) and MYH9/NMMHC-IIA and MYH10/NMMHC-IIB receptors . Beyond viral restriction, TMEFF1 may function as a tumor suppressor in brain cancers and plays a role in inhibiting NODAL and BMP signaling during neural patterning .

What is the cellular localization of TMEFF1?

TMEFF1 is localized to the cell membrane as a single-pass type I membrane protein . This membrane localization is essential for its function in preventing HSV-1 infection, as it acts at the cell surface to block viral entry mechanisms. Immunofluorescence studies using confocal microscopy have visualized TMEFF1 on the cell membrane, which can be co-stained with wheat germ agglutinin (WGA) for membrane visualization .

What are the common applications for TMEFF1 antibodies in research?

TMEFF1 antibodies are typically used in the following applications:

Researchers should verify the suitability of specific antibodies for their experimental design, as reactivity may vary between species (human, mouse, cynomolgus) .

How should TMEFF1 antibodies be stored and reconstituted for optimal performance?

For optimal performance of TMEFF1 antibodies:

• Store lyophilized antibodies at -20 to -70°C for up to 12 months from the date of receipt .

• For reconstitution, add sterile distilled water to achieve a final antibody concentration of 1 mg/mL. Gently shake to solubilize the protein completely without vortexing .

• After reconstitution, store at 2-8°C for up to 1 month or at -20 to -70°C for up to 6 months under sterile conditions .

• Avoid repeated freeze-thaw cycles as they can compromise antibody activity .

• Some formulations, such as the purified monoclonal antibody from Abcepta, are supplied in PBS at pH 6.0 without preservative .

What validation methods should be used to confirm TMEFF1 antibody specificity?

To validate TMEFF1 antibody specificity, researchers should:

• Perform side-by-side testing in TMEFF1-expressing cells versus TMEFF1-knockout cells (generated via CRISPR-Cas9) .

• Verify membrane localization through co-staining with membrane markers like wheat germ agglutinin (WGA) .

• Confirm that staining patterns match known tissue distribution (high in brain, lower in heart, placenta, and skeletal muscle) .

• Compare antibody performance across multiple detection methods (Western blot, immunofluorescence, flow cytometry).

• Include isotype controls to rule out non-specific binding.

Research indicates that TMEFF1 protein should be absent from the cell membrane of TMEFF1-KO neurons while being clearly detectable on the cell surface of wild-type neurons .

How can TMEFF1 antibodies be used to investigate HSV-1 restriction mechanisms?

For investigating TMEFF1's role in HSV-1 restriction:

• Compare viral replication rates in TMEFF1-knockout versus wild-type neurons at various time points post-infection. Studies show significantly higher viral replication in TMEFF1-KO neurons .

• Use TMEFF1 antibodies in co-immunoprecipitation experiments to detect interactions with viral entry proteins.

• Perform rescue experiments by reintroducing TMEFF1 expression in knockout cells and measuring the effect on viral susceptibility.

• Include TLR3-deficient and IFNAR1-deficient neurons as comparisons, as TMEFF1-deficient neurons show viral replication rates similar to these models .

• Test the effect of IFNβ pretreatment, which rescues the phenotype in TMEFF1-deficient and TLR3-deficient neurons but not in IFNAR1-deficient neurons .

How can TMEFF1 antibodies be utilized in co-immunoprecipitation studies of nodal signaling pathways?

For investigating TMEFF1's role in nodal signaling through co-immunoprecipitation:

• Use mild lysis conditions (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions.

• Consider that TMEFF1-Cripto interactions may be weaker than Cripto-ALK4 interactions, potentially requiring optimization of experimental conditions .

• Include positive controls (e.g., Cripto-ALK4 interaction) and negative controls (e.g., isotype-matched IgG) .

• Target the extracellular domain of TMEFF1, as research shows the cytoplasmic domain is dispensable for its nodal inhibitory activity .

• Investigate interactions with specific domains, particularly the Cripto-FRL1-Cryptic (CFC) domain in Cripto, which is essential for both ALK4 binding and TMEFF1 interaction .

Research has demonstrated that TMEFF1 selectively inhibits nodal but not activin signaling by binding directly to the Cripto coreceptor .

What are common challenges when working with TMEFF1 antibodies, and how can they be addressed?

Common challenges and solutions when working with TMEFF1 antibodies include:

How can researchers distinguish between TMEFF1 and related proteins?

To distinguish TMEFF1 from related proteins:

• Select antibodies that target unique epitopes not shared with related proteins.

• Perform parallel RT-qPCR analysis to confirm protein detection correlates with mRNA expression levels .

• Include TMEFF1-knockout controls to verify antibody specificity .

• When investigating related family members, compare expression patterns across tissues (TMEFF1 is predominantly expressed in brain) .

• Consider using multiple antibodies targeting different epitopes to confirm results.

How can TMEFF1 antibodies be applied to investigate its potential tumor suppressor role?

To investigate TMEFF1's potential tumor suppressor function:

• Use immunohistochemistry with TMEFF1 antibodies to compare expression levels between normal brain tissues and brain tumors. Studies have shown TMEFF1 is downregulated in brain tumors compared to control brain tissues .

• Perform correlative studies between TMEFF1 expression levels and tumor grade, progression, or patient outcomes.

• Investigate the effect of TMEFF1 restoration in tumor cell lines using functional assays for proliferation, migration, and invasion.

• Examine downstream signaling pathways affected by TMEFF1 expression, particularly those related to NODAL and BMP signaling .

• Develop tissue microarrays with matched normal/tumor samples to facilitate high-throughput analysis of TMEFF1 expression patterns.

What experimental approaches can investigate TMEFF1's developmental role in neural patterning?

For studying TMEFF1's role in neural development:

• Use TMEFF1 antibodies to track expression patterns during neural differentiation of pluripotent stem cells.

• Compare NODAL and BMP signaling markers in wild-type versus TMEFF1-knockout or TMEFF1-overexpressing neural progenitors.

• Perform rescue experiments with wild-type and mutant forms of Cripto to investigate the specificity of TMEFF1's inhibitory effect .

• Utilize TMEFF1 antibodies in tissue sections to map expression patterns during embryonic brain development.

• Implement time-course studies to correlate TMEFF1 expression with key developmental milestones.

Research indicates that TMEFF1 may inhibit NODAL and BMP signaling during neural patterning , suggesting an important developmental role that warrants further investigation.

How should researchers quantify and compare TMEFF1 expression levels across different experimental conditions?

For accurate quantification of TMEFF1 expression:

• Use standardized methods for protein extraction, particularly for membrane proteins.

• Include loading controls appropriate for membrane proteins (such as Na⁺/K⁺-ATPase) rather than cytoplasmic proteins like GAPDH.

• Perform parallel RT-qPCR analysis to correlate protein levels with mRNA expression .

• When comparing TMEFF1 levels across cell types, normalize to total membrane protein content rather than total cellular protein.

• Consider flow cytometry for quantitative cell-by-cell analysis of surface TMEFF1 expression.

What statistical approaches are most appropriate for analyzing TMEFF1 antibody-generated data in viral infection studies?

For statistical analysis of TMEFF1-related viral infection data:

• For viral replication studies, use repeated measures ANOVA to analyze time-course experiments comparing TMEFF1-deficient versus wild-type cells .

• Include appropriate post-hoc tests (e.g., Tukey's or Bonferroni) for multiple comparisons.

• For rescue experiments (e.g., with IFNβ treatment), use two-way ANOVA to assess the interaction between cell genotype and treatment .

• Consider non-parametric alternatives (e.g., Mann-Whitney U test) if data do not meet normality assumptions.

• Present data with appropriate error bars (standard deviation or standard error of the mean) and clearly state sample sizes and replication numbers.

How might TMEFF1 antibodies be used in developing antiviral approaches based on its HSV restriction mechanisms?

Potential research directions for TMEFF1-based antiviral approaches:

• Use TMEFF1 antibodies to identify the specific domains and amino acid residues involved in HSV-1 restriction.

• Screen for small molecules that mimic TMEFF1's interaction with viral receptors.

• Investigate whether soluble forms of TMEFF1's extracellular domain retain antiviral activity.

• Explore whether TMEFF1 restricts other neurotropic viruses beyond HSV-1 and HSV-2.

• Develop assays to monitor TMEFF1 expression changes during viral infection and neuroinflammation.

Research demonstrates that TMEFF1 is a key restriction factor for HSV-1 in cortical neurons, with its constitutively high abundance protecting these cells from infection .

What novel techniques might enhance the utility of TMEFF1 antibodies in studying protein-protein interactions?

Emerging techniques for studying TMEFF1 interactions:

• Proximity labeling approaches (BioID, APEX) to identify proteins in the vicinity of TMEFF1 at the cell membrane.

• Single-molecule imaging to visualize TMEFF1-Cripto interactions in real-time.

• Cryo-electron microscopy to determine the structural basis of TMEFF1's interaction with binding partners.

• CRISPR-Cas9 knock-in of tagged TMEFF1 for improved antibody-independent detection.

• Multiplex immunofluorescence to simultaneously visualize TMEFF1 and its interaction partners in tissue contexts.