TENM1 Antibody

What is TENM1 and what is its role in neuronal development?

TENM1 (Teneurin Transmembrane Protein 1) is a member of the teneurin family of transmembrane proteins involved in cell adhesion and neuronal development. It plays a crucial role in neuronal development, axon guidance, and synapse formation in the central nervous system. TENM1 is implicated in cell adhesion processes and interactions with other proteins, making it a key player in neural circuit formation and maintenance. Understanding its function is essential for unraveling the complexities of brain development and neuroplasticity, with potential implications for neurological disorders .

What is the molecular structure and localization of TENM1?

TENM1 is a large protein with a calculated molecular weight of approximately 305-306 kDa, consisting of 2732 amino acids. It is primarily localized in the cell membrane, nucleus, and cytoplasm. The protein contains several structural domains, including an N-terminal intracellular teneurin domain, EGF-like repeats, NHL (ncl-1, HT2A and lin-41) repeats, and YD (Tyr and Asp dipeptide) domains. The extracellular portion contains regions that are critical for protein-protein interactions while the intracellular domain may function in cellular signaling .

What types of TENM1 antibodies are available for research?

Several types of TENM1 antibodies are available, including:

Researchers should select antibodies based on their specific application requirements and target species .

What are the optimal conditions for Western blot detection of TENM1?

For Western blot detection of TENM1, the following protocol is recommended:

• Use freshly prepared tissue/cell lysates, particularly from brain tissue or neuronal cell lines (e.g., U-87MG, U-251MG, H69/H146 cells)

• Apply a dilution ratio of 1:500-1:2000 for most polyclonal TENM1 antibodies

• Due to the high molecular weight of TENM1 (305 kDa), use low-percentage (6-8%) SDS-PAGE gels and extend transfer time

• Positive controls should include human brain tissue lysate, mouse brain tissue, or rat brain tissue

• Blocking with 5% non-fat milk or BSA in TBST for 1 hour at room temperature typically provides optimal results

Special consideration should be given to the large size of the protein, which may require optimization of transfer conditions to ensure efficient detection .

How should immunohistochemistry protocols be optimized for TENM1 detection?

For optimal IHC detection of TENM1:

• Use antigen retrieval with TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0 may be used)

• Apply antibody dilutions between 1:50-1:500 depending on antibody sensitivity

• For FFPE tissue sections, more extended antigen retrieval times may be necessary due to the large size of the protein

• Human and mouse brain tissues serve as excellent positive controls

• Include negative controls (secondary antibody only) to ensure specificity

• For fluorescent detection (IF), use a dilution of approximately 1:50-1:200

• Counterstain with DAPI to visualize nuclei and provide context for membrane/cytoplasmic staining

How do I design experiments to study TENM1's role in neuronal development?

To effectively study TENM1's role in neuronal development:

• Use model systems such as primary neuronal cultures, neuronal cell lines, or animal models (Drosophila, zebrafish, or mice) as demonstrated in recent studies

• Implement knockdown approaches using RNAi (as shown in Drosophila studies) or CRISPR-Cas9 gene editing

• Analyze phenotypes through electrophysiological recordings to detect seizure-like behavior and altered neuronal firing patterns

• Employ immunofluorescence with TENM1 antibodies to track expression patterns during different developmental stages

• Consider co-immunoprecipitation experiments to identify interaction partners

• For in vivo studies, conditional knockout models may be preferable to avoid embryonic lethality if TENM1 is essential for development

How can I distinguish between TENM1 and other teneurin family members in my experiments?

Distinguishing between TENM1 and other teneurin family members requires careful consideration:

• Select antibodies raised against unique epitopes specific to TENM1 - particularly those targeting the N-terminal domains which show greater variability between family members

• Verify antibody specificity through western blotting in tissues known to express different teneurin proteins

• Consider using recombinant expressed domains as controls

• Implement siRNA knockdown of TENM1 as a negative control to confirm antibody specificity

• For gene expression studies, design PCR primers that span unique regions of TENM1 to avoid cross-reactivity

• When possible, use multiple antibodies targeting different epitopes of TENM1 to confirm results

Some commercial antibodies specifically note they are not expected to cross-react with other TENM family members .

What are the potential confounding factors when measuring TENM1 expression in pathological samples?

When analyzing TENM1 expression in pathological samples, researchers should be aware of:

• Post-translational modifications affecting antibody binding - TENM1 may be proteolytically cleaved with the intracellular domain translocating to the nucleus

• Variable expression across different brain regions - regional specificity should be considered when comparing normal vs. pathological samples

• Developmental stage-dependent expression - TENM1 is primarily expressed in the developing central nervous system

• Hypoxic conditions may affect TENM1 expression through hypomethylation of a CpG island in the TENM1 gene

• Inflammatory mediators like IL-6 can upregulate TENM1 through Stat3-mediated pathways, particularly in glioblastoma

• The large size of TENM1 protein makes it susceptible to degradation during sample preparation, potentially leading to inconsistent results

How can quantitative binding affinity measurements be applied to TENM1 antibody characterization?

For quantitative characterization of TENM1 antibody binding affinities:

• Implement advanced methodologies like Tite-Seq (Titration-Sequencing) for parallel measurement of antibody binding curves

• This approach overcomes confounding effects of antibody expression and stability in standard deep mutational scanning assays

• Binding titration curves can be established by:

  • Displaying antibodies on yeast cell surfaces

  • Incubating with fluorescently labeled antigen at multiple concentrations

  • Sorting cells based on fluorescence intensity

  • Plotting binding curves to determine affinity constants

• For TENM1-specific applications, consider adapting this method by using recombinant TENM1 domains as antigens

• Calculate KD values (dissociation constants) to quantitatively compare different antibody clones

What is the evidence linking TENM1 to childhood epileptic encephalopathy?

Recent research has identified TENM1 as a causative gene for childhood epileptic encephalopathy, particularly Lennox-Gastaut syndrome:

• Whole-exome sequencing of 235 unrelated cases revealed X-linked recessive variants in TENM1 in six cases

• Five hemizygous missense variants were identified: c.467A>G/p.Asp156Gly, c.503G>A/p.Cys168Tyr, c.638C>T/p.Ala213Val, c.3326C>T/p.Thr1109Met, and c.5246T>C/p.Val1756Ala

• All TENM1 hemizygous variants were inherited from asymptomatic mothers, consistent with an X-linked recessive inheritance pattern

• The variants showed domain-specific effects:

  • Three variants in the N-terminal intracellular teneurin domain were associated with refractory seizures

  • Three variants in non-functional regions achieved seizure-free status under combination therapy

• Functional studies in animal models (Drosophila and zebrafish) demonstrated that knockdown of TENM1 orthologs resulted in increased seizure-like behavior and increased firing of excitatory neurons

How is TENM1 involved in cancer biology, particularly in glioblastoma?

TENM1 has been implicated in glioblastoma (GBM) pathophysiology through multiple mechanisms:

• TENM1 is upregulated in glioblastoma cells through a Stat3-mediated pathway activated by IL-6, which is released by tumor-associated monocytes

• Hypoxic microenvironments regulate GBM tumor cell migration partly by inducing TENM1 through hypomethylation of a CpG island in the TENM1 gene

• As a transmembrane protein involved in cell adhesion, TENM1 may contribute to the invasive behavior of glioblastoma cells

• TENM1 is a direct target of the homeobox transcription factor EMX2, which has been implicated in cortical development and potentially in oncogenesis

• Expression studies using TENM1 antibodies have demonstrated elevated levels in GBM cell lines, including U-87MG and U-251MG

What are the latest methodological advances in studying protein-protein interactions involving TENM1?

Recent methodological advances for studying TENM1 protein interactions include:

• Advanced antibody-based techniques for mapping the sequence-affinity landscape of protein interactions:

  • Tite-Seq technology allows for measuring binding titration curves for thousands of variant proteins in parallel

  • These methods eliminate confounding effects of protein expression and stability in traditional assays

  • Can be adapted to yeast display systems compatible with TENM1 domains

• Combination of multiple detection methods to validate interactions:

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity ligation assays in tissue samples

  • FRET (Förster Resonance Energy Transfer) with fluorescently tagged proteins

• Structural studies leveraging cryo-electron microscopy to elucidate the three-dimensional architecture of TENM1 and its binding partners, which is particularly valuable for large proteins like TENM1 (305 kDa)

What are the emerging therapeutic implications of TENM1 research?

The growing understanding of TENM1 biology points to several therapeutic possibilities:

• For epileptic encephalopathies:

  • Domain-specific effects of TENM1 variants suggest potential for personalized treatment approaches

  • Patients with variants in non-functional regions showed better response to valproate and lamotrigine combination therapy

  • Development of therapies targeting TENM1-related neuronal hyperexcitability

• For glioblastoma:

  • Targeting the IL-6/Stat3/TENM1 pathway could potentially reduce tumor invasiveness

  • Hypoxia-induced TENM1 upregulation suggests combination approaches with anti-angiogenic therapies

  • Consideration of TENM1 as a biomarker for treatment stratification

• The development of specific antibodies against extracellular domains of TENM1 could potentially be used for targeted therapies in both neurological disorders and cancers where TENM1 is implicated

How might quantitative approaches improve our understanding of TENM1 function?

Advanced quantitative methods will enhance TENM1 research through:

• Implementation of high-throughput methods like Tite-Seq for:

  • Measuring binding affinities between TENM1 and potential interaction partners

  • Mapping functional domains within the large TENM1 protein

  • Understanding the impact of genetic variants on protein function

• Single-cell transcriptomic and proteomic analyses to:

  • Track TENM1 expression at cellular resolution during development

  • Identify cell populations most affected by TENM1 dysfunction in disease models

  • Correlate TENM1 levels with other molecular markers

• Computational modeling of TENM1 structure-function relationships to:

  • Predict the impact of disease-associated variants

  • Identify potential drug binding sites

  • Understand the molecular mechanisms underlying TENM1's role in cell adhesion and signaling

What are the challenges in developing next-generation TENM1 antibodies for research applications?

Developing improved TENM1 antibodies faces several challenges:

• The large size of TENM1 (305 kDa) presents difficulties in:

  • Producing full-length recombinant protein for immunization

  • Ensuring epitope accessibility in various applications

  • Maintaining protein stability during experimental procedures

• Domain-specific antibodies require:

  • Careful epitope selection to avoid cross-reactivity with other teneurin family members

  • Validation across multiple applications (WB, IHC, IF, IP)

  • Confirmation of specificity using knockout/knockdown controls

• Next-generation approaches may include:

  • Developing antibodies against post-translationally modified forms of TENM1

  • Creating application-optimized antibodies for specific research contexts

  • Producing antibodies capable of distinguishing between membrane-bound and cleaved nuclear forms of TENM1