TEKT1 Antibody

What is TEKT1 and what are its key molecular characteristics?

TEKT1 (tektin 1) is a protein with a calculated molecular weight of 48 kDa, though it is typically observed at 48-50 kDa in western blot applications. The protein is encoded by the TEKT1 gene (Gene ID: 83659) and has been primarily characterized for its role in ciliary structures. TEKT1 is a member of the tektin family, which contributes to the structural integrity of cilia and flagella. The human TEKT1 protein (UniProt ID: Q969V4) functions at both primary and motile cilia where it plays critical roles in ciliary assembly and motility .

What are the typical subcellular localizations of TEKT1 observed in immunostaining experiments?

TEKT1 demonstrates specific subcellular localization patterns that are important to consider when designing immunostaining protocols. In cycling cells, TEKT1 localizes at the centrosome, colocalized with γ-tubulin. In ciliated cells, it is present at the basal bodies of both primary and motile cilia and extends to the axoneme of motile cilia specifically in airway cells. Immunofluorescence experiments have shown that while TEKT1 is found at the base of primary cilia (colocalizing with basal body markers), it is absent from the axoneme of primary cilia. This distinct localization pattern is critical for accurate interpretation of staining results and requires proper antibody dilution (typically 1:10-1:100 for IF/ICC applications) .

Which species reactivity has been validated for commercially available TEKT1 antibodies?

Commercial TEKT1 antibodies have been validated for reactivity against human, mouse, and rat samples. These validations have been confirmed through multiple experimental approaches including Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF) applications. When designing experiments with new tissue types or species, preliminary validation is recommended as reactivity may vary across antibody sources. For example, Proteintech's antibody (18968-1-AP) has demonstrated positive WB detection in human brain tissue, human testis tissue, and rat testis tissue, while IHC has been validated specifically in human testis tissue .

What are the recommended antibody dilutions for different experimental applications with TEKT1 antibody?

The optimal dilution of TEKT1 antibody varies significantly depending on the application and should be empirically determined for each experimental system. Based on validated protocols, the following dilution ranges are recommended:

It is strongly recommended that researchers perform a titration of the antibody in their specific testing system to obtain optimal results, as signal strength can vary based on expression levels and sample preparation methods .

What antigen retrieval methods are recommended for immunohistochemical detection of TEKT1?

For optimal immunohistochemical detection of TEKT1, antigen retrieval with TE buffer at pH 9.0 is recommended as the primary method. Alternatively, citrate buffer at pH 6.0 may also be effective, though potentially with reduced sensitivity. The choice of antigen retrieval method is particularly important when working with formalin-fixed, paraffin-embedded tissues where protein cross-linking can mask epitopes. For tissues with low TEKT1 expression, the higher pH TE buffer (pH 9.0) typically provides better epitope retrieval and stronger signal. Researchers should compare both methods when establishing protocols for new tissue types to determine which provides optimal signal-to-noise ratio .

How can I validate the specificity of TEKT1 antibody detection in my experimental system?

Validating TEKT1 antibody specificity requires a multi-faceted approach. First, perform western blot analysis to confirm detection of a single band at the expected molecular weight (48-50 kDa). For definitive validation, include appropriate controls such as:

• Lysates from cells with TEKT1 knockdown (siRNA or CRISPR-based approaches)

• Competitive blocking with the immunizing peptide

• Testing in tissues known to express TEKT1 (testis and brain tissues show reliable expression)

• For transfection experiments, compare GFP-TEKT1 fusion detection with endogenous protein

Immunofluorescence validation should include colocalization studies with established centrosomal/basal body markers such as γ-tubulin, pericentrin, or centrin. The absence of staining in knockdown cells and proper subcellular localization (centrosomal in cycling cells, basal body in ciliated cells) provide strong evidence for antibody specificity .

What are the critical considerations when designing experiments to study TEKT1 localization in primary versus motile cilia?

Designing experiments to differentiate TEKT1 localization between primary and motile cilia requires careful consideration of cell types, markers, and imaging techniques. Key considerations include:

• Cell type selection: Use cell lines known to form primary cilia (RPE1, IMCD3 cells, fibroblasts) versus those with motile cilia (airway epithelial cells)

• Cilia induction: For primary cilia, serum starvation (24-48 hours) is typically required

• Co-staining markers:

  • Use acetylated tubulin to mark ciliary axonemes

  • Include γ-tubulin or pericentrin to mark basal bodies/centrosomes

  • For motile cilia, include additional markers like RSPH4A (radial spoke proteins)

• High-resolution imaging: Confocal or super-resolution microscopy is essential to distinguish basal body from axonemal localization

Research has demonstrated that TEKT1 localizes to the basal body in both primary and motile cilia, but extends to the axoneme only in motile cilia. This differential localization is functionally significant and may relate to TEKT1's role in ciliary motility versus sensory functions .

How should I design knockdown experiments to study TEKT1 function in ciliated cells?

Designing effective TEKT1 knockdown experiments requires careful planning to avoid off-target effects while achieving sufficient depletion. Based on published methodologies, consider the following approach:

• siRNA design: Target conserved regions of TEKT1 mRNA; use at least 2-3 different siRNA sequences to control for off-target effects

• Transfection optimization: For ciliated cells like RPE1, optimize transfection conditions to minimize toxicity while achieving >70% knockdown efficiency

• Knockdown verification: Confirm knockdown by both qRT-PCR and western blot (50% reduction in protein levels is typically sufficient to observe phenotypes)

• Timing considerations: Transfect cells before ciliation induction; typical protocol involves:

  • Day 1: Seed cells

  • Day 2: Transfect siRNA

  • Day 3: Induce ciliation by serum starvation

  • Day 5-6: Analyze phenotypes

When analyzing knockdown effects, quantify multiple parameters including cilia formation frequency (% ciliated cells), cilia length, and morphology. Research has shown that TEKT1 knockdown does not affect cilia formation but significantly reduces cilia length (2.2±0.67 μm vs 3.1±0.7 μm in controls), indicating its role as a positive regulator of cilium length .

What are common issues in TEKT1 antibody staining and how can they be resolved?

When working with TEKT1 antibodies, researchers commonly encounter several technical challenges that can be systematically addressed:

For tissues showing auto-fluorescence (particularly in IHC applications), include appropriate quenching steps or consider chromogenic detection alternatives. When staining for basal body localization, pre-extraction with detergents before fixation can sometimes improve visualization of centrosomal structures .

How should I interpret contradictory results between different TEKT1 antibodies?

When faced with contradictory results using different TEKT1 antibodies, a systematic analytical approach is essential:

• Compare epitope regions: Different antibodies may target distinct domains of TEKT1, which might be differentially accessible depending on protein interactions or conformational states

• Evaluate validation methods: Review how each antibody was validated (KO/KD controls, peptide competition, etc.)

• Consider isoform specificity: Determine if antibodies might recognize different TEKT1 isoforms or post-translationally modified forms

• Employ complementary techniques: Use multiple methods (IF, WB, IP) to build a consensus view of TEKT1 localization and function

• Genetic verification: Whenever possible, complement antibody studies with genetic approaches (GFP-tagged constructs, CRISPR-mediated tagging)

If discrepancies persist, design experiments that can clarify which results are more reliable, such as rescue experiments with wild-type TEKT1 in knockdown cells to verify phenotype specificity. Consider that seemingly contradictory results might reveal context-dependent properties of TEKT1 function or localization .

How can TEKT1 antibodies be used to investigate the functional impact of TEKT1 mutations identified in ciliopathy patients?

TEKT1 mutations have been implicated in ciliopathies, particularly those affecting motile cilia function. To investigate the functional consequences of patient-derived mutations:

• Immunolocalization studies: Compare wild-type versus mutant TEKT1 localization using antibodies against endogenous or tagged proteins. Research has shown that pathogenic variants (p.R244* and p.K311N) fail to localize to the centrosome, while non-pathogenic variants (p.R232Q) maintain normal localization

• Expression analysis: Quantify expression levels of mutant proteins, as some mutations lead to decreased protein stability (50% reduction observed with certain mutations)

• Functional rescue experiments: Test if wild-type TEKT1 expression can rescue phenotypes in patient-derived cells or model systems

• Structure-function analysis: Use domain-specific antibodies to determine which structural features are disrupted by specific mutations

For example, studies have demonstrated that the p.R244* TEKT1 truncation results in undetectable protein (likely due to nonsense-mediated decay), while the p.K311N missense variant produces stable protein that fails to properly localize to centrosomes and basal bodies. These differential effects provide insight into the molecular mechanisms underlying TEKT1-associated ciliopathies .

What methodological approaches can be used to study TEKT1 interactions with other ciliary proteins?

Investigating TEKT1's protein interaction network requires specialized methodologies optimized for ciliary and cytoskeletal proteins:

• Co-immunoprecipitation: Use TEKT1 antibodies for IP followed by mass spectrometry to identify novel interacting partners. Critical considerations include:

  • Use of mild detergents (0.5% NP-40 or 0.1% Triton X-100) to preserve interactions

  • Crosslinking steps for transient interactions

  • Validation of key interactions with reverse IP

• Proximity labeling approaches: Implement BioID or APEX2-based proximity labeling with TEKT1 fusions to identify neighboring proteins in the centrosomal/ciliary environment

• Two-hybrid screening: Use yeast or mammalian two-hybrid systems to test direct interactions with candidate partners

• Immunofluorescence co-localization: Perform super-resolution microscopy with TEKT1 antibodies and antibodies against known ciliary components to determine spatial relationships

• In vitro binding assays: Express recombinant TEKT1 domains to map specific interaction interfaces

When analyzing interaction data, consider the dynamic nature of ciliary assembly and the potential for cell-cycle dependent interactions. Focus particular attention on interactions with other tektin family members and with components of the ciliary microtubule structure .

How can TEKT1 antibodies be used to differentiate between primary and motile ciliopathies in research settings?

Distinguishing between primary and motile ciliopathies is a significant diagnostic challenge. TEKT1 antibodies can be valuable tools in this differentiation:

• Differential localization analysis: TEKT1 localizes to basal bodies in both primary and motile cilia but extends to the axoneme only in motile cilia. This differential pattern can be exploited to characterize ciliopathy types

• Patient sample analysis: Compare TEKT1 distribution in biopsy samples (typically nasal or bronchial) from patients with suspected ciliopathies

• Quantitative assessment: Measure the ratio of axonemal to basal body TEKT1 staining as a potential biomarker for motile cilia dysfunction

• Combined marker approach: Use TEKT1 antibodies alongside markers for:

  • Motile cilia (DNAH5, DNAI2)

  • Primary cilia (ARL13B, IFT88)

Research has shown that mutations in genes like WDR19 typically affect primary cilia, while TEKT1 mutations may predominantly impact motile cilia. In cases with combined ciliopathies (affecting both types), this dual-targeting approach with appropriate antibodies can provide valuable diagnostic insights. The case reported in the literature of a patient with compound heterozygous mutations in both WDR19 and TEKT1 demonstrated clinical features of both primary ciliopathies (renal, retinal, and skeletal involvement) and motile ciliopathies (recurrent lung infections and airway ciliary dyskinesia) .

What are the latest research findings on TEKT1 function in cilia, and how can TEKT1 antibodies contribute to advancing this knowledge?

Recent research has expanded our understanding of TEKT1's functions in ciliary biology, with several key findings:

• Regulation of cilium length: TEKT1 acts as a positive regulator of cilium length, with knockdown resulting in significantly shorter cilia (2.2±0.67 μm vs 3.1±0.7 μm in controls)

• Genetic interactions: Studies have revealed potential genetic interactions between TEKT1 and other ciliopathy genes like WDR19, suggesting coordinated functions in ciliary assembly and maintenance

• Evolutionary conservation: TEKT1 function appears conserved across species, with zebrafish knockdown experiments confirming its role in ciliary motility

To advance these research directions, TEKT1 antibodies can be employed in:

• High-throughput screening: Identify small molecules that alter TEKT1 localization or function as potential therapeutic agents

• Developmental studies: Track TEKT1 expression during ciliogenesis and organ development in model organisms

• Patient stratification: Develop immunostaining protocols to classify ciliopathy patients based on TEKT1 expression/localization patterns

• Structure-function analysis: Map functional domains through deletion constructs and domain-specific antibodies

As ciliopathies represent an expanding group of genetic disorders, TEKT1 antibodies will likely play an increasingly important role in both basic research and translational applications aimed at understanding and treating these conditions .