TENM1 Antibody, HRP conjugated

What is TENM1 and why is it important in neurodevelopmental research?

TENM1 (teneurin transmembrane protein 1) is a member of a family of four neuronal cell surface proteins homologous to the Drosophila pair-rule gene Ten-m. It is expressed primarily in the developing central nervous system and plays crucial roles in neuronal connectivity . TENM1 is particularly significant because:

• It may undergo proteolytic cleavage with the intracellular domain translocating to the nucleus

• It functions as a direct target of the homeobox transcription factor EMX2, which is important for area specification in the developing cortex

• It has been implicated in synaptic partner matching during neural development

• Recent studies have linked TENM1 variants to childhood epileptic encephalopathy

The expression pattern of TENM1 is highly specific, with strong signals detected in the mitral cells of the olfactory bulb, subpopulations of neurons in the hippocampus and piriform cortex, retinal ganglion cells, and neurons in the rotund nucleus and optic tectum . This specific expression pattern makes TENM1 a valuable marker for studying neural circuit formation.

What is the molecular structure and expected size of TENM1 protein?

TENM1 is a large transmembrane protein with several functional domains:

While the calculated molecular weight of TENM1 is approximately 305 kDa, it typically appears as a 280 kDa band in Western blot analyses . This discrepancy is common with large proteins due to factors such as post-translational modifications or protein folding affecting mobility during electrophoresis .

What are the key considerations when selecting a TENM1 antibody for research applications?

When selecting a TENM1 antibody for research, consider the following technical aspects:

• Specificity: Verify that the antibody is specific to TENM1 and doesn't cross-react with other teneurin family members (TENM2-4)

• Epitope location: Antibodies targeting different domains (intracellular vs. extracellular) may yield different results depending on proteolytic processing of TENM1

• Validated applications: Confirm that the antibody has been validated for your intended application (WB, IF, IHC)

• Species reactivity: Ensure compatibility with your experimental model (human, mouse, rat)

• Positive controls: Human brain tissue lysate is recommended as a positive control for TENM1 antibody validation

For optimal results in different applications, follow these dilution guidelines:

• Western blot: 1:500-1:2000

• Immunofluorescence: 1:50-1:200

What are the advantages of using HRP-conjugated antibodies for TENM1 detection?

HRP (horseradish peroxidase) conjugation offers several advantages for TENM1 detection in research applications:

• Enhanced sensitivity: HRP enzymatic amplification enhances signal detection compared to direct fluorescent labels

• Stable signal: HRP produces a stable chromogenic or chemiluminescent signal suitable for various detection methods

• Versatility: HRP-conjugated antibodies can be used across multiple applications including Western blot, ELISA, and immunohistochemistry

• Cost-effectiveness: HRP substrates are generally less expensive than fluorescent imaging systems

• Compatibility: HRP detection systems are compatible with standard laboratory equipment and do not require specialized fluorescence microscopes

For TENM1 detection specifically, HRP-conjugated antibodies are valuable because they can detect the protein even at low expression levels, which is important considering the tissue-specific expression pattern of TENM1 in neuronal subpopulations .

How should HRP-conjugated TENM1 antibodies be optimally stored and handled?

Proper storage and handling of HRP-conjugated TENM1 antibodies is crucial for maintaining their activity:

• Long-term storage: Store the lyophilized powder at or below -20°C, where it remains stable for at least one year

• After reconstitution: Store at 4°C for short-term use (up to one week)

• For prolonged storage after reconstitution: Add glycerol to a final concentration of 50% (v/v), make aliquots, and store at or below -20°C, where the solution is stable for approximately three months

• Avoid freeze/thaw cycles: Repeated freezing and thawing significantly reduces antibody activity

• Pre-use preparation: Briefly centrifuge protein conjugate solutions in a microcentrifuge before use and use only the supernatant to eliminate protein aggregates that may cause nonspecific background staining

• Working concentration: Reconstitute using PBS (pH 7.2) to yield a 1 mg/mL stock solution

What protocols are recommended for optimizing HRP-conjugated antibody performance in Western blot assays?

For optimal Western blot results with HRP-conjugated TENM1 antibodies:

• Sample preparation considerations:

  • Use fresh tissue lysates when possible

  • Human brain tissue lysate is recommended as a positive control

  • When working with TENM1, note that standard protein extraction methods may be suitable, but the protein's large size (280-305 kDa) requires special attention during sample preparation and gel separation

• Recommended protocol optimizations:

  • Use gradient gels (4-15%) to better resolve the large TENM1 protein (280 kDa)

  • Extend the transfer time for large proteins (typically 12-16 hours at low voltage)

  • Block membranes thoroughly to minimize background (5% non-fat dry milk or BSA in TBST, 1-2 hours at room temperature)

  • Start with a 1:1000 dilution for the HRP-conjugated TENM1 antibody and adjust based on signal strength

  • Include 0.05% Tween-20 in wash buffers to reduce non-specific binding

  • Consider using enhanced chemiluminescent substrates for detection due to the potentially low expression levels of TENM1 in some tissues

• Troubleshooting guidance:

  • If the observed band differs from the expected 280-305 kDa size, this may be due to post-translational modifications, alternative splicing, or proteolytic processing of TENM1

  • If multiple bands appear, verify if they represent different isoforms or processed forms of TENM1, as the protein is known to undergo proteolytic cleavage

How can TENM1 expression patterns be effectively visualized in the developing nervous system?

Visualizing TENM1 expression patterns in the developing nervous system requires specialized approaches:

• Immunohistochemistry/immunofluorescence protocol refinements:

  • Use perfusion fixation with 4% paraformaldehyde for optimal tissue preservation

  • Consider antigen retrieval methods to expose the TENM1 epitope (citrate buffer, pH 6.0)

  • Use a 1:50-1:100 dilution of HRP-conjugated TENM1 antibody for optimal staining

  • Include nuclear counterstains (DAPI) to better visualize cellular context

  • Use confocal microscopy for detailed localization studies

• Key brain regions for TENM1 expression analysis:
  Based on published research, prioritize examining these regions where TENM1 is highly expressed :

  • Mitral cells of the olfactory bulb

  • Hippocampus and piriform cortex

  • Retinal ganglion cells and cells in the inner nuclear layer

  • Rotund nucleus

  • Stratum griseum centrale of the optic tectum

  • Nucleus laminaris and nucleus magnocellularis

  • Cerebellum (particularly Purkinje cells)

• Developmental timeline considerations:
  TENM1 expression changes throughout development, so examine multiple developmental timepoints for comprehensive analysis .

What methodological approaches are recommended for studying TENM1's role in synaptic partner matching?

Recent research has identified TENM1 as a key player in synaptic partner matching . To study this function:

• Experimental models and systems:

  • Drosophila olfactory system provides a well-characterized model for studying teneurin function in synaptic matching

  • Vertebrate models should focus on regions with high TENM1 expression, such as the visual system connections between the retina and tectum

• Recommended methodological approaches:

  • Genetic manipulations: Use CRISPR/Cas9 to introduce specific TENM1 variants identified in human disorders

  • Co-immunoprecipitation: Identify TENM1 binding partners using HRP-conjugated TENM1 antibodies

  • Super-resolution microscopy: Visualize TENM1 localization at synapses using HRP-conjugated antibodies with tyramide signal amplification

  • Electrophysiology: Combine with TENM1 immunolabeling to correlate protein expression with functional connectivity

• Key signaling pathways to investigate:
  Based on recent findings, TENM1 signaling involves :

  • RhoGAP regulation

  • Rac1 GTPase activation

  • Local F-actin remodeling

  • Axon branch stabilization upon contact with partner dendrites

How can researchers approach TENM1 variant analysis in neurological disorders?

Recent studies have identified TENM1 variants in childhood epileptic encephalopathy . For researchers investigating TENM1's role in neurological disorders:

• Variant identification and characterization:
  Five hemizygous missense variants in TENM1 have been identified in childhood epilepsy cases :

  Variant	Protein Change	Domain Location	Clinical Presentation
  c.467A>G	p.Asp156Gly	N-terminal intracellular teneurin domain	Refractory seizures
  c.503G>A	p.Cys168Tyr	N-terminal intracellular teneurin domain	Refractory seizures
  c.638C>T	p.Ala213Val	N-terminal intracellular teneurin domain	Refractory seizures
  c.3326C>T	p.Thr1109Met	Extracellular region between EGF-like repeats and NHL repeats	Responsive to treatment
  c.5246T>C	p.Val1756Ala	Extracellular region between YD 5 and YD 6 domains	Responsive to treatment

• Experimental approaches for functional characterization:

  • Domain-specific antibodies: Use antibodies targeting different TENM1 domains to assess the impact of variants on protein expression and localization

  • In vitro assays: Develop cell-based assays to evaluate the effect of variants on TENM1 processing and nuclear translocation

  • Animal models: Generate knock-in models of specific variants to assess their impact on neurodevelopment and seizure susceptibility

• Correlation with clinical findings:

  • Variants in the N-terminal intracellular domain appear to correlate with more severe, treatment-resistant phenotypes

  • Extracellular domain variants may have milder clinical manifestations

How can researchers address the challenges of detecting TENM1 due to its large size and variable processing?

TENM1's large size (280-305 kDa) and potential for proteolytic processing present unique challenges:

• Optimized protein extraction:

  • Use lysis buffers containing protease inhibitors to prevent degradation

  • Consider non-denaturing conditions when studying protein-protein interactions

  • For membrane protein extraction, include 0.5-1% Triton X-100 or NP-40 in lysis buffers

• Electrophoretic separation strategies:

  • Use low percentage (6-8%) or gradient (4-15%) SDS-PAGE gels

  • Extend running time at lower voltage

  • Consider using specialized large-protein electrophoresis systems

• Detection of processed forms:

  • Use antibodies targeting different domains (N-terminal vs. C-terminal) to detect specific fragments

  • Compare results between reducing and non-reducing conditions

  • Perform parallel immunoprecipitation experiments with domain-specific antibodies

What are the recommended controls for validating TENM1 antibody specificity in different experimental contexts?

Proper validation of TENM1 antibody specificity is crucial:

• Positive controls:

  • Human brain tissue lysate is recommended as a primary positive control

  • Cell lines with verified TENM1 expression: U87-MG, U-251MG, HeLa for Western blot; C6, L929, U-2OS for immunofluorescence

• Negative controls:

  • TENM1 knockout/knockdown samples

  • Pre-absorption of the antibody with the immunizing peptide

  • Secondary antibody-only controls

  • Tissues known to lack TENM1 expression

• Specificity validation approaches:

  • Cross-reactivity testing against other TENM family members

  • Peptide competition assays

  • Multiple antibody approach (use antibodies from different sources or targeting different epitopes)

How can researchers optimize HRP-conjugated TENM1 antibody performance for co-localization studies with other neuronal markers?

For co-localization studies with HRP-conjugated TENM1 antibodies:

• Multiplexing strategies:

  • Use tyramide signal amplification (TSA) to convert HRP signal to a fluorescent readout compatible with multi-channel imaging

  • Perform sequential staining with HRP inactivation between rounds

  • Consider using nanobody-based detection systems for better penetration in thick tissues

• Compatible neuronal markers:
  Based on TENM1's expression pattern, consider co-staining with markers for:

  • Mitral cells (Tbx21)

  • Retinal ganglion cells (Brn3a)

  • Purkinje cells (Calbindin)

  • General neuronal markers (NeuN, MAP2, Tau)

• Protocol optimizations:

  • Test different fixation methods (PFA vs. methanol) for optimal epitope preservation

  • Optimize antigen retrieval conditions

  • Use appropriate blocking reagents to minimize background

  • Consider tissue clearing techniques for thick section imaging

What are the implications of TENM1 in cancer research and how can HRP-conjugated antibodies facilitate these studies?

Recent research has identified TENM1 as a potential marker for cancer progression, particularly in papillary thyroid cancer :

• Key findings in cancer research:

  • TENM1 expression is highly upregulated in papillary thyroid cancer tissues compared to benign thyroid tissues

  • TENM1 expression correlates with the classical subtype of papillary thyroid cancer, extrathyroidal invasion, BRAF V600E mutation, and advanced disease stage

  • Transcriptome analyses indicate differential TENM1 expression from stage I to stage IV in papillary thyroid cancer

• Experimental approaches for cancer studies:

  • Immunohistochemistry on tissue microarrays using HRP-conjugated TENM1 antibodies

  • Correlation of TENM1 expression with clinical parameters and patient outcomes

  • TENM1 knockdown/overexpression studies in cancer cell lines

• Potential research directions:

  • Investigation of TENM1 as a prognostic biomarker for thyroid cancer

  • Exploration of the mechanistic role of TENM1 in cancer progression

  • Development of TENM1-targeting therapeutic approaches

How can researchers effectively study TENM1 intracellular domain nuclear translocation?

TENM1 is known to undergo proteolytic cleavage with potential nuclear translocation of its intracellular domain :

• Experimental approaches for studying nuclear translocation:

  • Use domain-specific antibodies to track the intracellular domain

  • Perform subcellular fractionation with subsequent Western blot analysis

  • Employ live-cell imaging with fluorescently tagged TENM1 constructs

• Recommended protocol refinements:

  • For immunofluorescence: Use anti-TENM1 antibodies specific to the intracellular domain

  • For biochemical analysis: Carefully separate nuclear and membrane fractions

  • For quantification: Develop image analysis pipelines to quantify nuclear vs. membrane signal ratios

• Potential inducing signals to investigate:

  • Based on data from other transmembrane proteins with regulated intramembrane proteolysis:

    • Calcium signaling

    • Neuronal activity

    • Ligand binding

    • Developmental cues

What methodological approaches should be considered when investigating TENM1's interaction with the EMX2 transcription factor?

TENM1 is a direct target of the homeobox transcription factor EMX2 , suggesting important developmental regulatory mechanisms:

• Experimental approaches for studying transcriptional regulation:

  • Chromatin immunoprecipitation (ChIP) assays to confirm EMX2 binding to TENM1 regulatory regions

  • Luciferase reporter assays to quantify EMX2-dependent TENM1 transcription

  • CRISPR interference or activation to modulate EMX2 activity and assess effects on TENM1 expression

• Co-expression analysis approaches:

  • Double immunofluorescence staining for EMX2 and TENM1 during critical developmental periods

  • Single-cell RNA sequencing to identify cell populations with co-expression

  • In situ hybridization with fluorescent markers for both genes

• Functional interaction studies:

  • Genetic interaction studies in model organisms

  • Analysis of TENM1 expression in EMX2 mutant backgrounds

  • Investigation of phenotypic consequences of disrupting the EMX2-TENM1 regulatory axis