TENM1 Antibody, FITC conjugated

What is TENM1 and what is its functional significance in neurological research?

TENM1 (Teneurin transmembrane protein 1) is a member of a family of neuronal cell surface proteins homologous to the Drosophila pair-rule gene Ten-m. It is expressed primarily in the developing central nervous system and may function as a cellular signal transducer. The protein may undergo proteolytic cleavage with the intracellular domain translocating to the nucleus. TENM1 is a direct target of the homeobox transcription factor EMX2, which is important for area specification in the developing cortex .

Recent research has identified TENM1 as a causative gene in childhood epileptic encephalopathy, specifically Lennox-Gastaut syndrome. X-linked recessive variants in TENM1 have been found in six unrelated cases, with specific variants located in different protein domains associated with varying treatment outcomes. Notably, cases with variants in the N-terminal intracellular teneurin domain suffered from refractory seizures even under multiple antiepileptic drug treatments, while those with variants in non-functional regions achieved seizure-free status under combination therapy .

What are the key characteristics of TENM1 protein?

TENM1 has the following characteristics:

*Note: The actual band observed in Western blot may not match the expected size as protein mobility can be affected by various factors including post-translational modifications .

What is FITC conjugation and how does it benefit immunofluorescence studies?

FITC (Fluorescein Isothiocyanate) conjugation involves crosslinking a primary antibody with the FITC fluorophore using established protocols. This direct conjugation eliminates the need for secondary antibodies in immunofluorescence detection, simplifying workflows and reducing background signal .

FITC-conjugated antibodies allow for direct visualization of target proteins in immunofluorescence experiments. When using a fluorescence microscope equipped with an appropriate FITC filter, researchers can directly observe the localization of TENM1 within cellular structures without additional detection steps .

What precautions should be taken when working with FITC-conjugated antibodies?

When working with FITC-conjugated antibodies, researchers should:

• Avoid continuous exposure to light as this will cause the antibody to gradually lose its fluorescence

• Store the antibody at -20°C and avoid repeated freeze/thaw cycles

• Work in reduced lighting conditions during experimental procedures

• Consider that sodium azide (commonly used as a preservative in antibody solutions) may interfere with some applications and can form explosive metal azides

What applications are TENM1 antibodies suitable for?

Based on commercially available antibodies, TENM1 antibodies have been validated for:

A recommended positive control for TENM1 antibody testing is human brain tissue lysate .

What is the recommended protocol for immunofluorescence using FITC-conjugated antibodies?

While the search results don't provide a TENM1-specific FITC protocol, a standard immunofluorescence protocol for FITC-conjugated antibodies includes:

• Fix cells in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 minutes at room temperature

• Wash cells three times with PBS

• Permeabilize cells in PBS with 0.1% Triton X-100 for 10 minutes at room temperature

• Block with PBS containing 5% BSA, 5% goat serum, and 0.01% Triton X-100 for 30 minutes

• Dilute the FITC-conjugated primary antibody in IF buffer (PBS, 5% BSA, 0.01% Triton X-100) at the recommended dilution (typically 1:10-1:100 for immunofluorescence)

• Incubate cells with the diluted antibody overnight at 4°C in the dark

• Wash cells 2 × 5 minutes with PBS

• Observe cells with a fluorescence microscope equipped with a FITC filter

How can I validate the specificity of TENM1 antibodies?

To validate TENM1 antibody specificity, researchers should:

• Use appropriate positive controls (human brain tissue lysate is recommended)

• Include negative controls (such as TENM1 knockout cell lines, similar to the approach used for TMEM106B antibody validation)

• Perform side-by-side testing of antibody performance in Western blot using wild-type and knockout samples

• For immunoprecipitation validation, evaluate the antibody by detecting the target protein in extracts, immunodepleted extracts, and immunoprecipitates

• For immunofluorescence, consider a mosaic approach using cells with and without TENM1 expression

How do TENM1 variants correlate with epileptic encephalopathy phenotypes?

Research has identified five hemizygous missense variants in TENM1 in six unrelated cases of childhood epileptic encephalopathy (typically Lennox-Gastaut syndrome). The variants show distinct genotype-phenotype correlations:

All TENM1 hemizygous variants were inherited from asymptomatic mothers, consistent with an X-linked recessive inheritance pattern. The aggregate frequencies of these variants were significantly higher in the patient cohort than in male controls .

What methodological approaches can be used to study TENM1 expression in neurological development?

For studying TENM1 expression in neurological development, researchers can employ:

• Immunofluorescence with FITC-conjugated antibodies: To visualize protein localization in neuronal cell cultures or tissue sections

• Western blot analysis: To quantify protein expression levels across different developmental stages

• qPCR analysis: Similar to the approach used for TMEM123, using specific primers to detect TENM1 mRNA expression

  • Example primer design approach: Design primers that span exon-exon junctions to avoid genomic DNA amplification

  • Perform reactions with appropriate cycling conditions (e.g., 95°C for 10 min followed by 50 cycles of amplification)

• Knockout/knockdown models: To study the functional consequences of TENM1 deficiency in neurological development, similar to the Drosophila and zebrafish studies that showed seizure-like behavior

What are the key considerations when selecting between different TENM1 antibodies for specialized applications?

When selecting TENM1 antibodies, researchers should consider:

• Target epitope: Different antibodies target different regions of TENM1. The search results mention antibodies targeting AA 1-241 region and recombinant fusion protein of human TENM1

• Cross-reactivity: Verify whether the antibody cross-reacts with other TENM family members. Some TENM1 antibodies are not expected to cross-react with other members of the TENM family

• Species reactivity: Confirm reactivity with your species of interest (human, mouse, rat)

• Application validation: Ensure the antibody has been validated for your specific application (WB, IF, etc.)

• Conjugation needs: Determine whether direct conjugation (e.g., FITC) is beneficial for your experimental design

How can I optimize signal-to-noise ratio when using FITC-conjugated TENM1 antibodies?

To optimize signal-to-noise ratio:

• Optimize antibody dilution: While standard recommendations exist (1:10-1:100 for IF), the optimal dilution should be determined empirically for each application and sample type

• Improve blocking: Use a robust blocking solution (PBS containing 10% fetal bovine serum) for at least 20 minutes at room temperature before antibody incubation

• Reduce background: Ensure thorough washing between steps (at least 2 × 5 minutes with PBS)

• Optimize fixation: Different fixation methods may yield different results; 4% paraformaldehyde is standard but alternative methods may be explored

• Adjust exposure settings: When imaging, carefully optimize exposure time to maximize signal while minimizing background

Why might I observe a different molecular weight band for TENM1 than expected?

The observed molecular weight of TENM1 (280 kDa) differs from the calculated weight (305 kDa). This discrepancy is common in Western blotting and can be attributed to:

• Post-translational modifications affecting protein mobility

• Alternative splicing (several alternatively spliced transcript variants encoding different isoforms have been found for TENM1)

• Proteolytic processing (TENM1 may be proteolytically cleaved)

• Different running conditions affecting migration patterns

• The presence of different protein domains and their folding properties affecting migration

When unexpected band sizes are observed, it's important to verify specificity through additional controls .

How might TENM1 antibodies contribute to epilepsy research?

Given the recent identification of TENM1 variants in epileptic encephalopathy, FITC-conjugated TENM1 antibodies could be valuable tools for:

• Studying protein localization patterns in epileptic versus normal neuronal tissues

• Investigating the impact of disease-associated mutations on protein expression and localization

• Developing potential diagnostic markers for certain types of genetic epilepsy

• Screening potential therapeutic compounds that might modulate TENM1 function

• Exploring genotype-phenotype correlations observed in clinical cases, such as the differential treatment response based on mutation location

What are the emerging methods for studying TENM1 interactions with other proteins in the neuronal membrane?

Emerging methods for studying TENM1 protein interactions include:

• Proximity labeling combined with mass spectrometry to identify interaction partners

• Super-resolution microscopy with FITC-conjugated antibodies to visualize nanoscale protein organization

• CRISPR-based methods to tag endogenous TENM1 with fluorescent proteins for live-cell imaging

• Electrophysiological studies combined with immunofluorescence to correlate TENM1 expression with neuronal activity patterns

• Patient-derived induced pluripotent stem cells (iPSCs) differentiated into neurons to study mutation effects in a human neurodevelopmental context