TEKT2 Antibody

What is TEKT2 and why is it important in research?

TEKT2 (Tektin 2) is an evolutionarily conserved filament-forming protein primarily localized in flagella and cilia. It plays a critical role in the stability and structural complexity of axonemal microtubules. Research has demonstrated that TEKT2 is required for normal flagellum structure and function, with TEKT2-null sperm displaying flagellum bending and reduced motility, likely due to disruption of the dynein inner arm . TEKT2 is particularly important in reproductive biology research as it is essential for sperm motility and fertility studies.

What applications can TEKT2 antibodies be used for?

TEKT2 antibodies have been validated for multiple applications including:

• Western Blot (WB) at dilutions ranging from 1:500 to 1:6000

• Immunohistochemistry (IHC) at dilutions ranging from 1:20 to 1:200

• Immunofluorescence (IF)

• Immunoprecipitation (IP)

• Enzyme-Linked Immunosorbent Assay (ELISA)

Each application requires specific optimization of antibody concentration and protocol conditions for optimal results.

What species reactivity is typically observed with TEKT2 antibodies?

Commercial TEKT2 antibodies show confirmed reactivity with:

• Human

• Mouse

• Rat

Some antibodies have also been cited in publications showing reactivity with bovine samples . When working with other species, cross-reactivity testing is recommended prior to experimental use.

How should I optimize TEKT2 antibody dilution for my specific application?

Optimal dilution varies by application and specific antibody. Based on validated protocols:

It is strongly recommended to perform a dilution series to determine optimal concentration for your specific experimental system .

How can I confirm TEKT2 antibody specificity in my experiments?

Multiple approaches can verify antibody specificity:

• Molecular weight verification: TEKT2 has a calculated molecular weight of 50 kDa but is typically observed at 54 kDa in SDS-PAGE

• Positive control tissues: Use mouse or rat testis tissue and mouse brain tissue, which are known to express TEKT2

• Blocking peptide competition: Pre-incubate antibody with the immunizing peptide to confirm signal elimination

• TEKT2 knockout/knockdown samples: Compare antibody reactivity in wild-type versus TEKT2-depleted samples

• Multiple antibody verification: Use antibodies targeting different TEKT2 epitopes to confirm consistent localization patterns

How is TEKT2 localized within sperm flagella at the ultrastructural level?

Immunoelectron microscopy and confocal laser scanning microscopy have revealed that TEKT2 is not directly associated with axonemal tubulins as previously thought. Instead, TEKT2 is primarily localized at the periphery of outer dense fibers (ODFs) in the sperm flagellum. This localization pattern was confirmed through sequential extraction experiments, which showed that TEKT2 is only partially released when axonemal tubulins are completely extracted with SDS-EDTA . This suggests that TEKT2 functions as an ODF-affiliated molecule that contributes to flagellum stability and sperm motility rather than being a core component of the axoneme itself.

How can I investigate TEKT2's protein-protein interactions in my research?

Several methodological approaches have been validated for studying TEKT2's protein interactions:

• Yeast two-hybrid screening: This approach successfully identified TEKT2BP1 (Ccdc172) as a TEKT2-binding protein. The full-length TEKT2 cDNA can be used as bait for screening against a testis cDNA library

• Co-immunoprecipitation:

  • Transfect cells with tagged TEKT2 constructs (e.g., EGFP-TEKT2)

  • Immunoprecipitate with anti-TEKT2 antibody

  • Identify binding partners by mass spectrometry or Western blot

• GST pull-down assays:

  • Express recombinant TEKT2 as a GST-fusion protein

  • Incubate with tissue/cell lysates

  • Analyze bound proteins by SDS-PAGE and mass spectrometry

• In vitro binding assays with purified proteins to confirm direct interactions

What are the critical considerations when performing immunofluorescence with TEKT2 antibodies on sperm samples?

When conducting immunofluorescence on sperm samples:

• Fixation: 4% paraformaldehyde in PBS at 4°C for 1 hour provides optimal preservation while maintaining antibody epitope accessibility

• Permeabilization: Two approaches have been validated:

  • Standard approach: 0.1% Triton X-100 for 5 minutes

  • Enhanced extraction: 1% Triton X-100, 0.1% SDS, and 1% 2-mercaptoethanol for 10 minutes (for accessing deeply embedded epitopes)

• Blocking: PBS containing 5% nonfat milk effectively reduces background

• Primary antibody incubation: Typically 2 hours with anti-TEKT2 antibody diluted 1:200 in blocking buffer

• Secondary antibody: Cy3-conjugated or other fluorophore-conjugated anti-rabbit IgG

• Counterstaining: SYTOX Green (1:10,000) for DNA visualization can help identify sperm heads and provide spatial reference

• Controls: Include pre-immune serum controls and TEKT2-null samples when possible

What could cause multiple bands or unexpected molecular weights when using TEKT2 antibodies in Western blot?

Multiple bands or unexpected molecular weights may result from:

• Post-translational modifications: While TEKT2's calculated molecular weight is 50 kDa, it is typically observed at 54 kDa, suggesting potential modifications

• Splice variants: Multiple isoforms may be detected depending on tissue source and antibody epitope

• Protein degradation: Inadequate sample preparation or storage can lead to degradation products

• Cross-reactivity: Antibodies may detect other tektin family members (TEKT1-5) due to sequence homology

• Sample preparation issues: Incomplete denaturation or reduction can affect migration patterns

To address these issues:

• Include positive control tissues (mouse testis/brain)

• Use fresh samples with protease inhibitors

• Optimize sample preparation conditions

• Consider using antibodies targeting different epitopes for confirmation

How can I distinguish between specific and non-specific binding in TEKT2 IHC/IF experiments?

To differentiate specific from non-specific binding:

• Implement proper controls:

  • Pre-immune serum control at the same concentration as the primary antibody

  • Peptide competition by pre-incubating antibody with immunizing peptide

  • Omit primary antibody (secondary antibody only)

  • Include known positive tissue (e.g., human trachea, rat testis) and negative tissue

• Examine localization pattern:

  • Specific TEKT2 staining should be concentrated in flagella/cilia-containing structures

  • In sperm, staining should be primarily in the flagellum, not the head

  • In trachea, staining should be present in ciliated epithelial cells

• Optimize protocol:

  • Titrate antibody concentration

  • Adjust blocking conditions

  • Modify antigen retrieval methods

  • Consider using amplification systems for low-abundance targets

How do I interpret contradictory results between different TEKT2 detection methods?

When faced with contradictory results:

• Consider method-specific limitations:

  • WB detects denatured proteins and may miss conformational epitopes

  • IHC preserves tissue architecture but may have limited sensitivity

  • IF provides spatial information but may suffer from background issues

  • IP effectiveness depends on epitope accessibility in native conditions

• Evaluate antibody characteristics:

  • Epitope location (N-terminal, C-terminal, internal)

  • Polyclonal vs. monoclonal specificity

  • Host species and potential cross-reactivity

• Reconciliation approaches:

  • Use multiple antibodies targeting different epitopes

  • Implement genetic models (knockout/knockdown)

  • Combine biochemical and imaging approaches

  • Consider mass spectrometry for unbiased protein identification

How can TEKT2 antibodies be used to study male infertility mechanisms?

TEKT2 antibodies provide valuable tools for investigating male infertility through several approaches:

• Immunophenotyping sperm abnormalities:

  • Compare TEKT2 localization/expression between fertile and infertile individuals

  • Correlate TEKT2 distribution patterns with specific motility defects

  • Examine potential associations between TEKT2 abnormalities and specific infertility diagnoses

• Structure-function studies:

  • Combine TEKT2 staining with functional assessments (e.g., computer-assisted sperm analysis)

  • Correlate ultrastructural defects (by electron microscopy) with TEKT2 localization

  • Investigate relationships between TEKT2 and other flagellar proteins in patient samples

• Genetic studies:

  • Examine TEKT2 expression/localization in patients with specific genetic variants

  • Use TEKT2 antibodies to verify the impact of TEKT2 mutations on protein expression and localization

What novel interactions between TEKT2 and other flagellar proteins have been discovered?

Recent research has identified important TEKT2 protein interactions:

• TEKT2BP1/Ccdc172: This 36-kDa protein interacts with TEKT2 and localizes to the mitochondrial sheath in the middle piece of sperm flagella. The TEKT2-TEKT2BP1 complex may be involved in the structural linkage between the outer dense fibers (ODFs) and mitochondria in the middle piece of sperm flagella .

• TEKT2BP2: Also identified through yeast two-hybrid screening, this protein represents another TEKT2-interacting molecule that may contribute to flagellar architecture .

• Axonemal components: While TEKT2 was initially thought to associate directly with axonemal tubulins, more recent evidence suggests that it associates with the periphery of ODFs and may form structural linkages between ODFs and other flagellar components .

These interactions provide important insights into the molecular architecture of mammalian sperm flagella and potential mechanisms underlying sperm motility defects.

How can combining TEKT2 immunolabeling with advanced imaging techniques enhance our understanding of flagellar structure?

Integration of TEKT2 immunolabeling with cutting-edge imaging approaches offers several advantages:

• Super-resolution microscopy:

  • Techniques such as STORM, PALM, or STED can resolve TEKT2 localization beyond the diffraction limit

  • Enables visualization of TEKT2 distribution relative to other flagellar components with nanometer precision

  • Can reveal previously undetectable organizational patterns

• Correlative light and electron microscopy (CLEM):

  • Combines immunofluorescence detection of TEKT2 with electron microscopy of the same sample

  • Provides both molecular specificity and ultrastructural context

  • Particularly valuable for understanding TEKT2's relationship to axonemal and peri-axonemal structures

• Live cell imaging with fluorescently tagged TEKT2:

  • Monitor TEKT2 dynamics during flagellar assembly and function

  • Investigate turnover and transport of TEKT2 in living systems

  • Study TEKT2 behavior during sperm capacitation and hyperactivation

These advanced approaches can significantly enhance our understanding of TEKT2's role in flagellar architecture and function beyond what conventional immunolabeling techniques can achieve.

What are the critical controls for validating TEKT2 antibody specificity across different experimental systems?

To ensure robust validation across experimental systems:

Documentation of these controls is essential for publication quality data and experimental reproducibility.

How can I optimize sample preparation for consistent TEKT2 detection in different tissue types?

Tissue-specific optimization strategies include:

• For testis and sperm samples:

  • Fixation: 4% paraformaldehyde (PFA) or 3% PFA with 0.1% glutaraldehyde for ultrastructural studies

  • Processing: For sperm, attach to poly-L-lysine-coated slides after fixation

  • Permeabilization: Triton X-100 (0.1-1%) with optional SDS (0.1%) and 2-mercaptoethanol (1%) for enhanced extraction

• For ciliated epithelial tissues (trachea, lung, etc.):

  • Fixation: 10% neutral buffered formalin

  • Antigen retrieval: TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0 (alternative)

  • Sectioning: 4-5 μm sections optimal for IHC

• For embedding media selection:

  • Paraffin embedding: Standard for IHC applications

  • LR White resin: Preferred for immunoelectron microscopy

  • OCT compound: For frozen sections when antigen sensitivity is a concern

• Storage considerations:

  • Store sections at -20°C if not used immediately

  • Minimize freeze-thaw cycles of antibody solutions

  • Prepare fresh working dilutions for each experiment

Systematic optimization and documentation of these parameters will significantly enhance reproducibility across experiments.

How do polyclonal and monoclonal TEKT2 antibodies compare in research applications?

Most commercial TEKT2 antibodies currently available are polyclonal, such as the frequently cited 13518-1-AP from Proteintech, which has demonstrated utility across multiple applications and species .

What epitope considerations should inform TEKT2 antibody selection for specific research questions?

When selecting TEKT2 antibodies for specific research:

• Structural considerations:

  • N-terminal epitopes: May be more accessible in native protein but could be affected by potential modifications

  • C-terminal epitopes: Consider if potential truncated variants are of interest

  • Internal epitopes: May be masked in properly folded protein but reliable in denatured applications

• Experimental application alignment:

  • For protein interaction studies: Select antibodies with epitopes outside predicted interaction domains

  • For post-translational modification studies: Choose antibodies with epitopes distant from potential modification sites

  • For isoform-specific detection: Target unique regions that distinguish between variants

• Evolutionary conservation:

  • For cross-species studies: Target highly conserved regions (helpful for studies in non-model organisms)

  • For paralog-specific detection: Select epitopes unique to TEKT2 versus other tektin family members

The peptide RGKIKKATED has been successfully used as an immunogen for generating TEKT2BP1 antibodies , suggesting that similar approaches with TEKT2-specific peptides can generate highly specific antibodies.

How can TEKT2 antibodies complement genomic and transcriptomic approaches in reproductive biology?

Integrating TEKT2 antibody-based techniques with other omics approaches provides comprehensive insights:

• Validating genomic findings:

  • Confirm protein-level impact of TEKT2 genetic variants identified through whole-exome/genome sequencing

  • Assess if structural variants or regulatory region mutations affect TEKT2 protein expression or localization

  • Correlate genotype with cellular phenotype through immunolocalization

• Extending transcriptomic insights:

  • Verify if TEKT2 mRNA expression patterns correlate with protein levels across developmental stages

  • Determine if alternative splicing events detected by RNA-seq produce detectable protein isoforms

  • Investigate post-transcriptional regulation by comparing mRNA and protein expression patterns

• Multi-omics research design:

  • Use RNA-seq to identify co-expressed genes in TEKT2-positive cells

  • Follow with co-immunoprecipitation and proteomics to identify interacting protein networks

  • Validate key interactions through co-localization studies with TEKT2 antibodies

This integrated approach can provide mechanistic insights into flagellar assembly and function that would not be possible with any single methodology.

What methodological considerations are important when using TEKT2 antibodies for analysis of clinical specimens?

When analyzing clinical samples:

• Specimen collection and preservation:

  • Standardize fixation protocols (timing, buffer composition, temperature)

  • Consider flash-freezing aliquots for protein extraction alongside fixed samples

  • Document clinical metadata that may impact TEKT2 expression (age, medications, pathological conditions)

• Staining protocol standardization:

  • Develop quantitative scoring systems for TEKT2 immunoreactivity

  • Include reference standards in each batch

  • Consider automated staining platforms for consistency

• Clinical correlation approaches:

  • Establish clear criteria for normal versus abnormal TEKT2 patterns

  • Use digital pathology for objective quantification where possible

  • Implement blinded assessment by multiple observers

• Ethical and consent considerations:

  • Ensure appropriate IRB approval and patient consent for research use

  • Consider privacy implications when sharing immunofluorescence images

  • Follow best practices for biospecimen research reporting