CHTF18 Antibody

What is CHTF18 and why is it significant in research?

CHTF18 (Chromosome Transmission Fidelity Factor 18 homolog) is a protein essential for meiotic recombination and spermatogenesis. Research indicates that CHTF18 functions in DNA double-strand break repair and crossover formation during meiosis, making it crucial for maintaining fertility in males. Disruption of the Chtf18 gene in mice results in subfertility, abnormal spermatogenic cell morphology, and perturbed seminiferous tubule arrangement . CHTF18 antibodies are valuable tools for investigating these processes in reproductive biology and genetic research.

What experimental applications are validated for CHTF18 antibodies?

CHTF18 antibodies have been validated for multiple experimental applications including:

Data compiled from multiple antibody providers indicates reproducible results across these applications, particularly with human samples .

How should sample preparation be optimized for CHTF18 detection by Western blot?

For optimal CHTF18 detection in Western blot applications:

• Tissue lysis should be performed in buffer containing protease inhibitors to prevent degradation, as CHTF18 is susceptible to proteolytic cleavage.

• When analyzing CHTF18 expression in transfected versus non-transfected cells, significant differences in band intensity can be observed at approximately 107-110 kDa .

• For detection of endogenous CHTF18, HeLa and Y79 cell lysates have demonstrated reliable signal .

• Antibody concentrations between 0.5-1.0 μg/ml typically yield optimal signal-to-noise ratios for Western blot applications .

Incomplete sample denaturation may result in higher molecular weight aggregates, compromising experimental interpretation.

What are the recommended protocols for immunohistochemical detection of CHTF18?

For immunohistochemical applications, the following methodological steps are critical:

• Antigen retrieval is essential - use TE buffer at pH 9.0 for optimal results, though citrate buffer at pH 6.0 can serve as an alternative .

• Antibody dilutions between 1:20-1:200 should be tested to determine optimal staining conditions for specific tissue types.

• CHTF18 expression has been successfully detected in human medulloblastoma tissue, providing a positive control reference .

• Nuclear staining pattern is expected, consistent with CHTF18's role in chromosome transmission fidelity.

When assessing spermatogenic tissues, careful attention to fixation protocols is required as over-fixation may mask epitopes in chromatin-associated proteins.

How can CHTF18 antibodies be utilized to investigate meiotic recombination defects?

For investigating meiotic recombination using CHTF18 antibodies:

• Co-immunostaining with CHTF18 antibodies and markers of DNA double-strand breaks (γH2AX) or recombination nodules (RAD51) can reveal persistence of repair intermediates.

• In normal meiosis, γH2AX staining decreases during prophase I as DSBs are repaired until it is confined to the sex body, while in Chtf18-null spermatocytes, γH2AX persists longer .

• RAD51 foci quantification in immunofluorescence microscopy (mean numbers per nucleus: 10.27 in wild-type versus 19.95 in Chtf18-null spermatocytes, p<0.0001) provides a quantitative measure of recombination defects .

• Combining CHTF18 staining with synaptonemal complex proteins like SYCP1 and SYCP2 allows assessment of homologous chromosome pairing during meiosis.

This multi-marker approach enables detailed investigation of the temporal sequence of recombination events and where CHTF18 functions in the process.

What experimental approaches can distinguish between pre-meiotic and meiotic functions of CHTF18?

To differentiate between pre-meiotic and meiotic functions:

• Employ conditional knockout models with stage-specific Cre-recombinase expression, similar to the TNAP-Cre system used to generate germ cell-specific Chtf18 deletion .

• Temporal analysis of spermatogenesis stages using synchronized cultures or developmental timeline studies.

• Complementation experiments with wild-type and mutant CHTF18 constructs can identify specific functional domains required for different stages.

• Analysis of CHTF18 interaction partners through co-immunoprecipitation followed by mass spectrometry at different developmental stages.

Research has demonstrated that germ cell-specific deletion of Chtf18 produces phenotypes indistinguishable from complete knockout, suggesting cell-autonomous function in germ cells rather than somatic effects .

How can researchers validate CHTF18 antibody specificity for their experimental system?

To validate CHTF18 antibody specificity:

• Compare staining patterns across multiple antibodies targeting different epitopes of CHTF18 (N-terminal, middle region, and C-terminal).

• Use positive controls such as HeLa cells which reliably express CHTF18 .

• Include genetic controls: Compare wild-type versus Chtf18 knockout tissues or cells (as confirmed by Western blot analysis showing absence of CHTF18 protein in knockout samples ).

• For recombinant protein systems, compare transfected versus non-transfected lysates, expecting a band at approximately 107-110 kDa in transfected samples only .

Caution is advised when interpreting results with single antibodies without proper validation controls.

What are the most common issues in CHTF18 antibody applications and how can they be addressed?

Common issues and solutions include:

How might CHTF18 antibodies contribute to understanding cancer biology?

CHTF18's role in chromosome transmission fidelity suggests potential implications in genomic stability and cancer:

• Immunohistochemical profiling of CHTF18 expression across different cancer types may reveal correlations with genomic instability phenotypes.

• The Human Protein Atlas project has catalogued CHTF18 expression in 17 different forms of human cancer, with potential correlations between expression levels and patient survival .

• Combined chromatin immunoprecipitation and sequencing (ChIP-seq) using CHTF18 antibodies could map genomic regions where CHTF18 operates in cancer cells versus normal cells.

• Multiplexed immunofluorescence combining CHTF18 with DNA repair markers may identify defective repair pathways in specific cancer subtypes.

Preliminary data suggests CHTF18 antibodies can detect the protein in cancer tissues including medulloblastoma , opening avenues for investigating its role in cancer progression.

What experimental approaches might elucidate CHTF18's protein interaction network during meiosis?

To map CHTF18's protein interaction network:

• Proximity-dependent biotin identification (BioID) coupled with CHTF18 antibodies for pulldown verification can identify proximal proteins in living cells.

• Co-immunoprecipitation with CHTF18 antibodies followed by mass spectrometry at different stages of meiosis.

• Yeast two-hybrid screening validated with co-immunoprecipitation using CHTF18 antibodies.

• FRET (Förster Resonance Energy Transfer) or FLIM (Fluorescence-Lifetime Imaging Microscopy) using fluorescently labeled CHTF18 antibodies to confirm direct protein interactions in situ.

Understanding CHTF18's interaction with proteins like RAD51, SYCP1, and SYCP2 during meiotic recombination would provide mechanistic insights into its role in ensuring proper chromosome segregation and fertility .

What epitope differences exist among commercial CHTF18 antibodies and how might they affect experimental outcomes?

Different commercial CHTF18 antibodies target distinct epitopes:

Epitope accessibility may vary depending on protein conformation, interaction partners, or post-translational modifications. For studies of protein complexes, antibodies targeting different regions may yield different results, necessitating validation with multiple antibodies .

How should researchers design experiments to study CHTF18's role in DNA damage response pathways?

For investigating CHTF18 in DNA damage response:

• Establish baseline CHTF18 expression and localization patterns before inducing DNA damage.

• Apply specific DNA damaging agents (e.g., ionizing radiation for double-strand breaks, UV for pyrimidine dimers, hydroxyurea for replication stress).

• Track CHTF18 dynamics using time-course immunofluorescence with co-staining for γH2AX to mark damage sites.

• Compare CHTF18 recruitment kinetics with known DNA repair factors such as RAD51.

• Design functional recovery assays (e.g., FRAP - Fluorescence Recovery After Photobleaching) to measure CHTF18 turnover at damage sites.

Previous research showing persistent γH2AX and RAD51 foci in Chtf18-null spermatocytes suggests CHTF18 functions in the double-strand break repair pathway, providing a foundation for further mechanistic studies .

How should researchers interpret discrepancies in CHTF18 localization between immunofluorescence and biochemical fractionation studies?

When facing discrepancies between techniques:

• Consider fixation artifacts - paraformaldehyde fixation may alter epitope accessibility in highly condensed chromatin structures.

• Evaluate extraction conditions - biochemical fractionation may disrupt protein complexes or release chromatin-bound CHTF18.

• Assess antibody accessibility - nuclear proteins may require permeabilization optimization to allow antibody penetration.

• Validate with complementary approaches - GFP-tagged CHTF18 localization in live cells compared with fixed-cell immunofluorescence.

• Perform chromatin immunoprecipitation to directly assess chromatin association.

Careful controls and multiple methodological approaches are needed to resolve discrepancies and establish accurate subcellular localization.

What methodological considerations are important when analyzing CHTF18 expression across different tissue types?

For cross-tissue expression analysis:

• Normalize protein loading using multiple housekeeping controls appropriate for each tissue type.

• Account for tissue-specific post-translational modifications that might affect antibody recognition.

• Consider cell-type heterogeneity within tissues - CHTF18 may be expressed in specific cell populations.

• In reproductive tissues, account for stage-specific expression during spermatogenesis.

• Use consistent fixation and antigen retrieval methods across all tissue samples.

CHTF18's expression throughout the male germline suggests tissue-specific functions , requiring careful experimental design to accurately compare expression levels across diverse tissue types.

What guidelines should researchers follow when using CHTF18 antibodies in studies with potential clinical implications?

For studies with potential clinical relevance:

• Antibody validation must meet stringent criteria, including confirmation with multiple antibodies targeting different epitopes.

• Include appropriate positive and negative controls for each experiment.

• Verify antibody lot-to-lot consistency through standardized validation protocols.

• Maintain detailed records of antibody source, catalog number, lot number, and validation data.

• Consider pre-registering experimental protocols to enhance reproducibility.

• When developing diagnostic applications, establish clear sensitivity and specificity parameters.

Given CHTF18's potential involvement in fertility and cancer, responsible reporting of experimental findings is essential to avoid misinterpretation in clinical contexts.

How can researchers ensure reproducibility when studying rare cell populations that express CHTF18?

To ensure reproducibility with rare cell populations:

• Implement standardized isolation protocols with clearly defined cell surface markers.

• Use flow cytometry sorting followed by validation of purity by immunostaining.

• Increase biological replicates to account for variability in rare populations.

• Consider single-cell approaches to avoid population averaging effects.

• Document detailed methodological parameters, including antibody concentrations, incubation times, and washing conditions.

• Validate findings across multiple experimental models or patient samples.

When studying CHTF18 in specific spermatogenic cell populations, careful staging and isolation techniques are critical for obtaining reproducible results.

How might phospho-specific CHTF18 antibodies contribute to understanding its cell cycle regulation?

Phospho-specific antibodies would enable:

• Mapping of CHTF18 phosphorylation sites during different cell cycle phases.

• Identification of kinases responsible for CHTF18 regulation.

• Temporal correlation between phosphorylation events and CHTF18 function in chromosome transmission.

• Investigation of how phosphorylation affects CHTF18's interaction with its binding partners.

• Potential therapeutic targeting of specific phosphorylation sites in disease contexts.

Since CHTF18 functions in meiotic recombination and chromosome transmission, its activity is likely regulated by post-translational modifications at specific cell cycle stages , making phospho-specific antibodies valuable research tools.

What novel applications of CHTF18 antibodies might emerge in the study of age-related fertility decline?

Emerging applications may include:

• Comparative immunohistochemistry of CHTF18 expression and localization in young versus aged testes.

• Analysis of CHTF18-dependent recombination events in oocytes at different maternal ages.

• Investigation of CHTF18 protein stability and turnover in aging reproductive cells.

• Assessment of CHTF18's interaction with age-affected DNA repair pathways.

• Correlation studies between CHTF18 expression/function and aneuploidy rates in aged gametes.