TCTE1 Antibody

What is TCTE1 and what are its known functions in cellular processes?

TCTE1, also known as DRC5, FAP155, and D6S46 in humans, is an evolutionarily conserved axonemal protein present in organisms from Chlamydomonas to mammals . It functions as a component of the nexin-dynein regulatory complex (N-DRC), which coordinates dynein arm activity in motile cilia and flagella . The protein is approximately 498 amino acids in length and contains a leucine-rich repeat domain at the C-terminus .

TCTE1's primary function appears to be in flagellar motility, particularly in sperm. Knockout studies in mice have demonstrated that TCTE1 is essential for male fertility, as Tcte1-null male mice are sterile due to impaired sperm motility . The protein plays a critical role in energy metabolism and structural integrity of sperm flagella, with its absence leading to complex metabolic disruptions affecting sperm function .

What types of TCTE1 antibodies are available for research applications?

Several types of TCTE1 antibodies are available for research purposes, including:

• Polyclonal antibodies targeting different regions of TCTE1:

  • C-terminal region antibodies (AA 285-501)

  • Middle region antibodies

  • N-terminal region antibodies (AA 1-300)

  • Specific epitope antibodies (AA 254-303, AA 336-385)

• Antibodies with different conjugations:

  • Unconjugated antibodies

  • HRP-conjugated antibodies

  • FITC-conjugated antibodies

  • Biotin-conjugated antibodies

Most commercially available TCTE1 antibodies are rabbit polyclonal antibodies validated for Western blot applications, though some are also suitable for ELISA, immunohistochemistry, and immunofluorescence .

What species reactivity is available for TCTE1 antibodies?

TCTE1 antibodies show reactivity across multiple species, reflecting the evolutionary conservation of the protein. Available antibodies demonstrate reactivity to:

• Human TCTE1

• Mouse TCTE1

• Rat TCTE1

• Guinea pig TCTE1

• Horse TCTE1

• Pig TCTE1

• Rabbit TCTE1

• Zebrafish (Danio rerio) TCTE1

The high predicted reactivity percentages (Human: 100%, Mouse: 100%, Rat: 100%, Horse: 100%, Pig: 100%, Rabbit: 92%, Guinea Pig: 86%, Zebrafish: 79%) indicate the strong conservation of epitopes across species . This cross-reactivity makes these antibodies valuable tools for comparative studies across different model organisms.

What are the primary applications for TCTE1 antibodies in research?

• Immunofluorescence staining to localize TCTE1 along sperm flagella

• Immunohistochemistry to detect TCTE1 in tissue sections

• ELISA for quantitative protein detection

• Co-immunoprecipitation studies to investigate protein-protein interactions within the N-DRC complex

These applications allow researchers to investigate TCTE1's expression, localization, and interactions in various experimental contexts related to male fertility and ciliary function.

How can TCTE1 antibodies be optimized for detecting protein in sperm samples?

Detecting TCTE1 in sperm samples presents specific challenges due to the protein's biochemical properties. Research indicates that TCTE1 is present in a Triton-resistant, SDS-soluble pool, making it difficult to extract using standard protocols . To optimize TCTE1 detection in sperm samples:

• Sample preparation: Use SDS-containing extraction buffers rather than milder detergents like Triton X-100, as studies show TCTE1 is only soluble in SDS-containing buffers .

• Fractionation approach: When studying localization, separate sperm heads from tails before Western blot analysis. This approach has successfully demonstrated TCTE1's presence in the tail fraction of spermatozoa .

• Immunofluorescence optimization: For immunofluorescence detection along the flagellum, proper fixation and permeabilization are critical. Paraformaldehyde fixation followed by Triton X-100 permeabilization has been successful in visualizing TCTE1 localization along the axoneme .

• Epitope consideration: Choose antibodies targeting the C-terminal region (containing leucine-rich repeats) for higher specificity, as this region contains characteristic epitopes of TCTE1 .

What experimental approaches can be used to investigate TCTE1's interaction with other N-DRC components?

Investigating TCTE1's interactions with other N-DRC components requires specialized approaches due to the complex nature of the axonemal structure. Based on published research, the following methods are recommended:

• Heterologous expression systems: Since direct co-immunoprecipitation from sperm is challenging due to TCTE1's solubility issues, express TCTE1 and potential interaction partners in cell culture systems. Studies have successfully used GFP-tagged TCTE1 co-transfected with FLAG-tagged DRC components in cell culture to identify interactions .

• Yeast two-hybrid screening: This approach can be used to screen for potential protein-protein interactions in a controlled system, bypassing the solubility challenges of native sperm proteins.

• Proximity labeling approaches: BioID or APEX2-based proximity labeling can identify proteins in close proximity to TCTE1 in vivo, potentially revealing interactions not detectable by co-immunoprecipitation.

• Cross-linking mass spectrometry: This technique can identify protein-protein interactions by cross-linking proteins in close proximity before mass spectrometric analysis.

Previous research has identified interactions between TCTE1 (DRC5) and several other DRC components, including DRC3, DRC6 (FBXL13), and DRC7 (CCDC135) , providing a foundation for further interaction studies.

How do proteomics findings from TCTE1 knockout models inform antibody-based experimental design?

Proteomic analysis of Tcte1-null versus wild-type spermatozoa has revealed significant differences that can inform antibody-based experimental design. Key considerations include:

• Differential protein expression: Proteomic studies have identified 446 differentially expressed proteins in Tcte1-null spermatozoa, with 398 down-regulated and only 49 up-regulated proteins . This suggests that TCTE1 loss affects numerous pathways that could be targeted in antibody-based studies.

• Metabolic pathway focus: Glycolytic enzymes are particularly affected in Tcte1-null sperm, with 12 glycolytic enzymes showing significantly reduced expression . Researchers should consider using antibodies against these enzymes alongside TCTE1 antibodies to investigate metabolic consequences of TCTE1 dysfunction.

• N-DRC component stability: Proteomic data suggests that TCTE1 does not regulate or stabilize other N-DRC components in mice . Therefore, antibody studies investigating other N-DRC components in TCTE1-deficient models may still yield detectable signals, allowing for studies of structural changes in the absence of TCTE1.

• ATP metabolism investigation: Since TCTE1 appears to affect energy metabolism, experimental designs that combine TCTE1 antibody localization with ATP level measurements can provide insights into the spatial relationship between TCTE1 localization and energy production in sperm flagella .

What considerations should be addressed when using TCTE1 antibodies for comparative studies across species?

When using TCTE1 antibodies for comparative studies across species, researchers should address several important considerations:

How can TCTE1 antibodies be used to investigate male infertility mechanisms?

TCTE1 antibodies can be valuable tools for investigating male infertility mechanisms, particularly in cases involving impaired sperm motility. Research strategies include:

• Clinical sample analysis: Use TCTE1 antibodies to assess protein expression and localization in sperm samples from infertile men, particularly those with conditions such as dysplasia of fibrous sheath (DFS) or multiple morphological abnormalities in the sperm flagella (MMAF) .

• Correlation with genetic findings: Combine antibody-based protein detection with genetic analysis of TCTE1 mutations in infertile patients to establish genotype-phenotype correlations. Recent research has identified mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males .

• Pathway investigation: Use TCTE1 antibodies alongside antibodies against proteins in relevant pathways identified from knockout studies, such as glycolysis enzymes and endoplasmic reticulum processing proteins, to investigate the molecular networks affected in infertility cases .

• Structure-function analysis: Employ immunofluorescence with TCTE1 antibodies to correlate protein localization patterns with flagellar ultrastructural abnormalities and motility parameters.

• Model system validation: Use TCTE1 antibodies to validate findings from mouse models in human clinical samples. The Tcte1 knockout mouse serves as a valuable model for male infertility research, showing complex effects on the testicular molecular network .

What controls should be included when using TCTE1 antibodies in research?

Proper controls are essential when using TCTE1 antibodies to ensure result validity. Recommended controls include:

• Negative controls:

  • Tissue/cells known not to express TCTE1 (e.g., non-testicular tissues for most applications)

  • Samples from Tcte1 knockout animals when available

  • Isotype control antibodies to assess non-specific binding

  • Primary antibody omission controls

• Positive controls:

  • Wild-type testis tissue or sperm samples

  • Samples from tagged-TCTE1 models (such as the Tcte1-FLAG mouse models described in the literature)

  • Recombinant TCTE1 protein (for Western blot applications)

• Validation controls:

  • Multiple antibodies targeting different epitopes to confirm specificity

  • Peptide competition assays to confirm binding specificity

  • siRNA or CRISPR knockdown samples to confirm signal reduction

Research has shown that commercial antibodies for TCTE1 have had reliability issues, with multiple attempts to generate TCTE1 antibodies failing to show promising results . Researchers have addressed this by creating epitope-tagged TCTE1 proteins in mice (Tcte1-FLAG), which can serve as excellent controls for antibody specificity testing .

What are the best fixation and sample preparation protocols for TCTE1 immunodetection?

Optimal fixation and sample preparation protocols for TCTE1 immunodetection depend on the specific application:

• For Western blotting:

  • Use SDS-containing extraction buffers, as TCTE1 is found in a Triton-resistant, SDS-soluble pool

  • For sperm samples, separate heads and tails before extraction to enrich for TCTE1 in the tail fraction

  • Standard SDS-PAGE loading buffers with reducing agents are appropriate

• For immunofluorescence staining:

  • Paraformaldehyde fixation (typically 4%) followed by Triton X-100 permeabilization has been successfully used to detect TCTE1 along sperm flagella

  • Methanol fixation may be considered as an alternative that combines fixation and permeabilization

  • For testis sections, consider antigen retrieval methods if initial staining appears weak

• For tissue immunohistochemistry:

  • Standard formalin fixation and paraffin embedding procedures are likely suitable

  • Antigen retrieval (heat-induced in citrate buffer) may be necessary to expose epitopes

  • Consider testing both frozen and paraffin-embedded sections to determine optimal preservation of the epitope

• For co-immunoprecipitation:

  • Due to solubility challenges, consider alternative approaches such as heterologous expression systems

  • If attempting direct IP from sperm, use stringent extraction conditions with SDS, followed by dilution to reduce SDS concentration before immunoprecipitation

What are the current limitations and future directions for TCTE1 antibody research?

Current limitations in TCTE1 antibody research include:

• Antibody reliability: Commercial antibodies have shown variable reliability, with multiple attempts to generate specific TCTE1 antibodies failing to show promising results . Researchers have addressed this by creating epitope-tagged TCTE1 proteins in mice, but this approach is not feasible for all research contexts, particularly clinical studies.

• Solubility challenges: TCTE1's biochemical properties (Triton-resistant, SDS-soluble) create challenges for certain applications like co-immunoprecipitation from native tissues .

• Limited functional information: While localization and knockout phenotypes are established, the precise molecular function of TCTE1 within the N-DRC remains incompletely characterized.

Future directions for TCTE1 antibody research include:

• Improved antibody development: Creation of more specific monoclonal antibodies targeting highly conserved epitopes of TCTE1 to enhance detection reliability across applications and species.

• Clinical correlations: Application of validated TCTE1 antibodies to study clinical samples from infertile men, particularly those with flagellar abnormalities, to establish the prevalence of TCTE1-related pathologies.

• Structural studies: Combining antibody labeling with high-resolution microscopy techniques like super-resolution microscopy or cryo-electron tomography to better understand TCTE1's position and function within the N-DRC.

• Therapeutic potential: Using antibodies to validate TCTE1 as a potential diagnostic marker or therapeutic target for specific forms of male infertility.

• Broader ciliopathy research: Expanding TCTE1 antibody applications to investigate potential roles in other ciliated tissues beyond sperm, given expression in tissues like the choroid plexus and Fallopian tube .