TEX11 Antibody

What is TEX11 and why is it important in reproductive research?

TEX11 is an X-chromosome-linked gene expressed specifically in germ cells that plays a crucial role in male fertility. It regulates homologous chromosomal synapsis and the repair of DNA double-strand breaks during meiosis . The importance of TEX11 in reproductive research stems from its association with male infertility, particularly non-obstructive azoospermia (NOA), where mutations in TEX11 account for approximately 1% of azoospermic men and up to 15% of azoospermia patients with meiotic arrest . Research with TEX11 antibodies enables the investigation of meiotic processes critical for proper spermatogenesis and can provide insights into the molecular basis of male infertility.

What are the recommended applications for TEX11 antibodies?

TEX11 antibodies are primarily used for:

• Immunohistochemistry (IHC): For detection of TEX11 in testicular sections to investigate its expression pattern during spermatogenesis .

• Western blotting (WB): For detection and quantification of TEX11 protein in tissue or cell lysates .

• ELISA: For quantitative analysis of TEX11 protein levels .

• Immunofluorescence (IF): For co-localization studies with other meiotic proteins .

For IHC applications, optimal dilutions typically range from 1:30 to 1:150, while for ELISA applications, dilutions of 1:5000 to 1:10000 are recommended .

What is the optimal sample preparation protocol for TEX11 immunohistochemistry?

Based on published methodologies, the following protocol is recommended for TEX11 IHC:

• Fixation: Formalin-fixed, paraffin-embedded tissue sections are commonly used.

• Deparaffinization: Heat slides at 60°C for 10 minutes, followed by xylene and ethanol rehydration series .

• Antigen retrieval: Use citrate buffer (pH 6.0) with heat-induced epitope retrieval .

• Blocking steps:

  • Inactivate endogenous peroxidase with 0.3% H₂O₂ solution

  • Block with 2.5% normal horse serum for 30 minutes at room temperature

• Primary antibody incubation: Apply TEX11 antibody at appropriate dilution (1:30-1:150) and incubate for 1.5 hours at 37°C .

• Detection: Use appropriate secondary antibody and DAB (3,3'-diaminobenzidine) visualization system, followed by hematoxylin counterstaining .

What controls should be included when working with TEX11 antibodies?

To ensure reliable results when working with TEX11 antibodies, include:

• Positive control: Normal testicular tissue with known TEX11 expression (typically human or mouse, depending on antibody specificity) .

• Negative control: Either:

  • Primary antibody omission

  • Non-immune serum of the same species as the primary antibody

  • Tissues known to lack TEX11 expression (non-reproductive tissues)

• Internal control: For comparative studies, include wild-type and TEX11-mutant tissues when available .

• Specificity validation: Consider including TEX11 knockout/knockdown samples where possible .

How can TEX11 antibodies be used to study meiotic arrest mechanisms?

TEX11 antibodies are valuable tools for investigating meiotic arrest mechanisms:

• Comparative analysis: Analyze TEX11 expression patterns in normal versus arrested spermatogenesis using IHC to identify the precise stage of meiotic arrest .

• Co-localization studies: Perform dual immunofluorescence with TEX11 antibodies and other meiotic markers (e.g., SYCP2, γH2AX) to assess synaptonemal complex formation and DNA damage repair mechanisms .

• TEX11 mutation analysis:

  • Use TEX11 antibodies targeting different epitopes to evaluate expression of mutant TEX11 proteins

  • Correlate antibody staining patterns with specific mutations to understand structure-function relationships

• Downstream signaling investigation: Combine TEX11 immunostaining with markers of apoptosis to establish links between TEX11 dysfunction and spermatocyte death .

• Quantitative assessment: Measure TEX11 protein levels in relation to meiotic progression to establish threshold levels required for normal spermatogenesis .

What methodological approaches are recommended for studying TEX11 interactions with other meiotic proteins?

To investigate TEX11 interactions with other meiotic proteins:

• Co-immunoprecipitation (Co-IP):

  • Use TEX11 antibodies for pull-down experiments in testicular lysates

  • Identify interacting partners through mass spectrometry or western blotting for known meiotic proteins

• Proximity ligation assay (PLA):

  • Combine TEX11 antibodies with antibodies against suspected interacting partners

  • Visualize protein-protein interactions in situ with subcellular resolution

• Immunofluorescence co-localization:

  • Use TEX11 antibodies in combination with antibodies against synaptonemal complex proteins (e.g., SYCP2)

  • Analyze co-localization during different stages of meiotic prophase I

• GST pull-down validation:

  • Express recombinant TEX11 protein domains to identify specific interaction regions

  • Validate interactions identified through other methods

• Yeast two-hybrid screening:

  • Use TEX11 as bait to identify novel interacting partners

  • Validate interactions using antibody-based techniques

How can TEX11 antibodies help distinguish between different types of azoospermia?

TEX11 antibodies can serve as diagnostic tools to distinguish between different types of azoospermia:

• Immunohistochemical analysis of testicular biopsies:

  • In non-obstructive azoospermia with TEX11 mutations: TEX11 expression may be absent, reduced, or abnormally localized

  • In meiotic arrest: TEX11 might be present in spermatogonia and early spermatocytes but absent in later stages

  • In obstructive azoospermia: Normal TEX11 expression pattern through all stages of spermatogenesis

• Quantitative assessment:

  • Measure TEX11 protein levels using calibrated immunostaining or western blotting

  • Correlate expression levels with specific types of spermatogenic failure

• Pattern analysis:

  • TEX11 is normally localized to the cytoplasm and nuclei of spermatogonia and early spermatocytes

  • Altered localization patterns may indicate specific types of meiotic dysfunction

• Combined marker approach:

  • Use TEX11 antibodies in conjunction with other stage-specific markers of spermatogenesis

  • Create a diagnostic algorithm based on expression patterns of multiple proteins

How do different TEX11 mutations affect antibody binding and epitope recognition?

Different TEX11 mutations can significantly impact antibody binding and epitope recognition:

• Frameshift mutations (e.g., c.151_154del p.D51fs) :

  • Antibodies targeting epitopes downstream of the frameshift will typically fail to detect the truncated protein

  • Antibodies targeting upstream epitopes may detect truncated protein with altered localization

• Deletion mutations (e.g., deletion of exons 9-11 in mice or 10-12 in humans) :

  • Antibodies targeting the deleted region will not bind

  • Antibodies targeting other regions can detect the truncated protein, as demonstrated in the Tex11^Ex9-11del/Y mouse model

• Missense mutations:

  • May cause conformational changes affecting epitope accessibility

  • Different antibodies targeting the same protein may show discrepant results depending on epitope location relative to the mutation

• Recommended approach:

  • Use multiple antibodies targeting different regions of TEX11

  • Select antibodies based on known mutation sites in your research subjects

  • For example, with exon 9-11 deletions, use C-terminal antibodies as demonstrated in mouse models

What are the optimal conditions for detecting TEX11 in different stages of spermatogenesis?

Optimal detection of TEX11 across different stages of spermatogenesis requires specific methodological considerations:

• Fixation optimization:

  • For early spermatogenic stages: 4% paraformaldehyde with shorter fixation times (4-6 hours)

  • For later stages: Bouin's fixative may provide better morphological preservation

• Antigen retrieval adjustment:

  • Early stages: Standard citrate buffer (pH 6.0) with moderate heating

  • Later stages: Consider EDTA buffer (pH 8.0) for enhanced epitope exposure

• Antibody selection:

  • For spermatogonia/early spermatocytes: Antibodies targeting N-terminal regions

  • For later stages: Antibodies recognizing C-terminal regions may be more effective

• Dilution optimization:

  • Stage-specific titration may be necessary

  • Generally, use lower dilutions (1:30-1:50) for later stages of spermatogenesis

• Detection system selection:

  • For co-localization studies: Fluorescent secondary antibodies

  • For quantitative analysis: HRP/DAB systems with standardized development times

• Counterstaining consideration:

  • Brief hematoxylin counterstaining (5 seconds) preserves specific signal while providing structural context

What species cross-reactivity should be considered when selecting TEX11 antibodies?

When selecting TEX11 antibodies, consider the following species cross-reactivity factors:

Table 1: TEX11 Antibody Species Reactivity From Available Research Data

Key considerations:

• TEX11 has approximately 75% homology between human and mouse at the mRNA level, with 86% coverage

• The protein alignment shows differences particularly in the first exons and the last exon

• When working with animal models, select antibodies validated for that specific species

• For comparative studies across species, choose antibodies targeting highly conserved regions

• Validate species cross-reactivity experimentally even when claimed by the manufacturer

How can TEX11 antibodies be used in studying cisplatin resistance in testicular germ cell tumors?

TEX11 antibodies can be valuable tools for investigating cisplatin resistance in testicular germ cell tumors (TGCTs):

• Expression analysis in resistant vs. sensitive cells:

  • Use western blotting and immunocytochemistry to quantify TEX11 expression

  • Evidence suggests TEX11 upregulation in cisplatin-resistant TGCT cell cultures

• Mechanistic studies:

  • Combine TEX11 immunostaining with DNA damage markers (γH2AX)

  • TEX11 silencing increases γH2AX-positive cells under cisplatin treatment, suggesting a role in DNA damage response

• Apoptosis investigation:

  • Use TEX11 antibodies alongside apoptotic markers (cleaved PARP1)

  • TEX11 appears to inhibit cisplatin-induced apoptosis in resistant cells

• In vivo tumor models:

  • Apply TEX11 immunohistochemistry to xenograft tissues

  • Correlate TEX11 expression with tumor response to cisplatin

• Therapeutic target validation:

  • Monitor TEX11 levels following experimental treatments

  • Use as a biomarker for treatment response in combination therapy approaches

• Recommended methodology:

  • For cell cultures: Use TEX11 antibodies at 1:100 dilution for western blotting

  • For tissue sections: Optimize antigen retrieval with citrate buffer pH 6.0

  • Include appropriate cisplatin-sensitive controls for comparison

What are common technical challenges when using TEX11 antibodies and how can they be addressed?

Researchers commonly encounter several challenges when working with TEX11 antibodies:

• Weak or absent signal:

  • Problem: Insufficient antigen retrieval or epitope masking

  • Solution: Extend antigen retrieval time or try alternative buffers (citrate pH 6.0 vs. EDTA pH 8.0)

  • Problem: Antibody concentration too low

  • Solution: Titrate antibody, starting with manufacturer's recommended dilution and adjusting as needed (1:30-1:150 for IHC)

• High background:

  • Problem: Insufficient blocking or non-specific binding

  • Solution: Increase blocking time (30+ minutes) and concentration (2.5-5% serum)

  • Problem: Secondary antibody cross-reactivity

  • Solution: Use species-specific secondary antibodies and include additional blocking steps

• Variable staining across samples:

  • Problem: Inconsistent fixation

  • Solution: Standardize fixation protocols (time, temperature, solution)

  • Problem: Tissue heterogeneity

  • Solution: Include larger sample areas and multiple sections for analysis

• Discrepant results between applications:

  • Problem: Epitope accessibility differs between techniques

  • Solution: Use different antibodies for different applications or optimize protocols for each technique

  • Problem: Buffer incompatibility

  • Solution: Verify compatibility of buffers with specific applications (PBS with 0.05% sodium azide and 40% glycerol for storage)

• Irreproducible results:

  • Problem: Antibody lot variation

  • Solution: Record lot numbers and test new lots against previous results

  • Problem: Protocol drift

  • Solution: Maintain detailed protocols with exact timing, temperatures, and reagent preparations

How can researchers validate the specificity of TEX11 antibodies?

Proper validation of TEX11 antibody specificity is crucial for reliable research outcomes:

• Genetic models validation:

  • Compare staining between wild-type and TEX11 knockout/knockdown models

  • Test antibodies in tissues from patients with known TEX11 mutations

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be significantly reduced or eliminated if specific

• Multiple antibody approach:

  • Use antibodies targeting different TEX11 epitopes

  • Concordant results increase confidence in specificity

• Western blot validation:

  • Confirm single band of expected molecular weight

  • For truncated proteins, verify appropriate size shift

• Recombinant protein controls:

  • Test antibody against recombinant TEX11 protein

  • Include both full-length and variant forms where relevant

• siRNA knockdown verification:

  • Compare staining between control and TEX11-specific siRNA treated samples

  • Document reduction in signal corresponding to knockdown efficiency

• Mass spectrometry confirmation:

  • Perform immunoprecipitation followed by mass spectrometry

  • Confirm TEX11 as the predominant recovered protein

How should immunohistochemical TEX11 expression patterns be interpreted in testicular biopsies?

Proper interpretation of TEX11 immunostaining in testicular biopsies requires understanding of normal expression patterns and potential alterations:

• Normal expression pattern:

  • TEX11 is primarily localized to the cytoplasm and nuclei of spermatogonia and early spermatocytes

  • Positive signal appears as brown precipitate following DAB visualization

  • Expression is typically observed at the periphery of certain seminiferous tubules

• Interpretation in non-obstructive azoospermia:

  • Complete absence of TEX11 staining may indicate null mutations

  • Abnormal localization may suggest missense mutations affecting protein trafficking

  • Reduced staining intensity might reflect hypomorphic alleles

• Analysis in meiotic arrest:

  • TEX11 may be detected in spermatogonia and early spermatocytes but absent in later stages

  • Compare with stage-specific markers to determine precise arrest point

• Quantitative assessment approaches:

  • Score percentage of TEX11-positive tubules

  • Evaluate staining intensity using standardized scales (0-3+)

  • Consider automated image analysis for objective quantification

• Comparison with normal controls:

  • Always include age-matched control tissue

  • Note physiological variations in expression levels

• Documentation standards:

  • Capture images at multiple magnifications (40x, 100x, 400x)

  • Include representative areas showing both positive and negative tubules

How do TEX11 protein levels correlate with genome-wide recombination rates?

Research has revealed important relationships between TEX11 protein levels and genome-wide recombination rates:

• Dosage-dependent effects:

  • TEX11 protein levels modulate genome-wide recombination rates in both sexes

  • Threshold levels of TEX11 are required for normal spermatogenesis

• Sex-specific differences:

  • TEX11 influences recombination rates differently in males versus females

  • These differences may contribute to known sexual dimorphism in recombination patterns

• Quantification methodology:

  • For accurate correlation, use quantitative western blotting with recombinant protein standards

  • Normalize TEX11 expression to appropriate housekeeping proteins (β-actin)

• Recombination markers:

  • Correlate TEX11 levels with established recombination markers (MLH1 foci)

  • Quantify crossover events using cytological approaches

• Genetic variation consideration:

  • TEX11 alleles affecting expression levels or amino acid substitutions may contribute to variations in recombination rates among individuals

  • Statistical analysis should account for genetic background effects

• Experimental design recommendations:

  • Include multiple TEX11 gene dosage models

  • Analyze both intra- and inter-individual variations

  • Consider tissue-specific and temporal regulation factors