FAM71F1 Antibody

What is FAM71F1 and what is its biological function?

FAM71F1, also known as GARI-L1 (Golgi-associated Rab2B interactor-like 1), is a protein that binds to RAB2A and RAB2B, which are small GTPases involved in vesicle transport and membrane trafficking. The primary biological function of FAM71F1 is to regulate acrosome formation during spermatogenesis. Research has shown that FAM71F1 forms a complex with active RAB2A/B to suppress excessive vesicle trafficking during acrosome development. This regulation is essential for normal sperm head morphology and male fertility. In FAM71F1-deficient mice, the acrosome becomes abnormally expanded at the round spermatid stage, likely due to enhanced vesicle trafficking, leading to male infertility .

What are the key specifications of commercially available FAM71F1 antibodies?

FAM71F1 antibodies are available from multiple manufacturers with various specifications for research use. A typical example is the polyclonal antibody from Proteintech (27984-1-AP), which has the following characteristics:

This antibody targets the protein encoded by the human FAM71F1 gene (Gene ID: 84691) . For research applications, it's important to validate the antibody specificity in your experimental system before proceeding with detailed studies.

In which tissues is FAM71F1 predominantly expressed?

FAM71F1 is predominantly expressed in testicular tissue. According to PCR analyses conducted in adult mice, both FAM71F1 and its paralog FAM71F2 show strong expression in the testis. Temporally, expression begins around postnatal day 21, corresponding to the onset of spermiogenesis. Single-cell RNA-sequencing data reveals that FAM71F1 expression dramatically increases at the mid-round spermatid stage (steps 4-6) of spermatogenesis . This expression pattern aligns with the protein's functional role in acrosome formation.

What are the optimal methods for detecting FAM71F1 in testicular tissue samples?

When investigating FAM71F1 in testicular tissue, researchers should consider multiple detection approaches:

• Immunofluorescence: For visualizing FAM71F1 localization during spermatogenesis, use paraffin-embedded or frozen testicular sections. Fix tissue with 4% paraformaldehyde, and apply the FAM71F1 antibody at optimized dilutions (typically 1:100-1:500). Co-staining with acrosomal markers (such as acrosin) and Golgi markers can help elucidate the protein's dynamics during acrosome formation.

• Western Blotting: For quantitative analysis, prepare testicular protein extracts using RIPA buffer supplemented with protease inhibitors. Use 20-40 μg of protein per lane, separate by SDS-PAGE, and transfer to PVDF membranes. The recommended antibody dilution for Western blotting is 1:1000-1:8000 .

• RT-PCR: For expression analysis, extract RNA from testicular tissue and synthesize cDNA. FAM71F1 expression can be detected from postnatal day 21 in mice, coinciding with the onset of spermiogenesis .

• Single-cell RNA sequencing: For detailed expression profiling across different stages of spermatogenesis. This approach has revealed that FAM71F1 expression significantly increases during the mid-round spermatid stage .

How can researchers differentiate between FAM71F1 and its paralogs in experimental systems?

Differentiating between FAM71F1 and its paralogs (particularly FAM71F2) requires careful experimental design:

• Antibody selection: Choose antibodies raised against unique epitopes of FAM71F1 that do not cross-react with paralogs. Validate antibody specificity using knockout models or siRNA knockdown approaches.

• Gene-specific primers: Design PCR primers that specifically amplify FAM71F1 but not its paralogs. Sequence verification of amplicons is essential to confirm specificity.

• Phenotypic analysis: Utilize the distinct phenotypic effects of FAM71F1 versus FAM71F2 mutations. For example, while both FAM71F1 and FAM71F2 mutations affect acrosome morphology, FAM71F1-mutant mice show more severe phenotypes, including impaired acrosome reaction even with Ca²⁺ ionophore treatment, which is not observed in FAM71F2-mutant mice .

• Protein-protein interaction studies: Leverage differential binding profiles, as FAM71F1 binds to both RAB2A and RAB2B, which might differ from the binding profiles of its paralogs .

What approaches can be used to investigate the FAM71F1-RAB2A/B interaction in vitro?

To investigate the molecular interaction between FAM71F1 and RAB2A/B proteins, consider these methodological approaches:

• Co-immunoprecipitation: Use anti-FAM71F1 antibody to pull down protein complexes from testicular lysates, followed by Western blotting for RAB2A and RAB2B. This technique has successfully demonstrated that FAM71F1 binds to both RAB2A and RAB2B in testicular tissue .

• GST pull-down assays: Express GST-tagged RAB2A/B (in both GTP-bound and GDP-bound states) and incubate with testicular lysates or recombinant FAM71F1. This approach can confirm that FAM71F1 preferentially binds to the GTP-bound active form of RAB2A/B .

• Yeast two-hybrid screening: This can identify specific domains mediating the FAM71F1-RAB2A/B interaction.

• FRET or BiFC analyses: These live-cell imaging techniques can visualize the FAM71F1-RAB2A/B interaction in real-time within cellular contexts.

• Surface plasmon resonance: This provides quantitative binding parameters (association/dissociation constants) for the interaction between purified FAM71F1 and RAB2A/B proteins.

How can researchers evaluate FAM71F1 function in acrosome biogenesis?

To evaluate FAM71F1's role in acrosome biogenesis, implement these experimental strategies:

• Knockout/knockdown models: Generate FAM71F1-deficient mice or use siRNA approaches in cultured cells. Analyze acrosome morphology at different stages of spermatogenesis using electron microscopy and acrosomal markers.

• Transmission electron microscopy (TEM): Abnormal swelling of the acrosome becomes apparent around step 5 in FAM71F1-deficient mice, with the phenotype persisting through mature spermatozoa .

• Immunofluorescence tracking: Use stage-specific markers to track acrosome formation. In FAM71F1-mutant mice, the acrosome appears normal until step 9 but shows swelling around steps 10-11 .

• Acrosomal protein localization: Examine the distribution of acrosomal proteins (e.g., IZUMO1) to assess acrosome integrity. IZUMO1 localization is impaired in FAM71F1-mutant spermatozoa .

• Functional assays: Evaluate acrosome reaction using calcium ionophore A23187 and monitor the release of acrosomal contents. FAM71F1-mutant spermatozoa show impaired acrosome reaction even with ionophore treatment .

What are the common issues when using FAM71F1 antibodies in Western blotting?

When using FAM71F1 antibodies for Western blotting, researchers may encounter several technical challenges:

• Non-specific bands: This may occur due to cross-reactivity with FAM71F1 paralogs or related proteins. Solution: Increase antibody dilution (e.g., 1:5000-1:8000), use more stringent washing conditions, and include additional blocking agents.

• Weak signal: FAM71F1 may be expressed at low levels in certain tissues or developmental stages. Solution: Increase protein loading (40-60 μg), enhance detection systems (e.g., use high-sensitivity ECL reagents), or concentrate the protein using immunoprecipitation before Western blotting.

• Unexpected molecular weight: The observed molecular weight for FAM71F1 is approximately 40 kDa , but post-translational modifications may alter migration. Solution: Include positive controls with verified FAM71F1 expression and consider using different gel concentrations to optimize separation.

• Sample preparation issues: Protein degradation during extraction may affect detection. Solution: Use fresh tissue samples, maintain cold conditions throughout extraction, and include multiple protease inhibitors in lysis buffers.

How can researchers address contradictory results in FAM71F1 expression studies?

When encountering contradictory results regarding FAM71F1 expression or function:

• Validate antibody specificity: Confirm antibody specificity using knockout/knockdown models or peptide competition assays. Multiple antibodies targeting different epitopes should yield consistent results.

• Consider developmental timing: FAM71F1 expression varies significantly during spermatogenesis, with peak expression at the mid-round spermatid stage . Inconsistencies may arise from analyzing different developmental timepoints.

• Evaluate detection methods: Different techniques (Western blot, immunofluorescence, PCR) have varying sensitivities. Compare results across multiple methods for comprehensive analysis.

• Assess biological context: Expression patterns may differ between species, strains, or under various physiological conditions. Clearly define the experimental system and avoid direct comparisons across disparate models.

• Examine tissue heterogeneity: In mixed cell populations, bulk analysis may mask cell-specific expression patterns. Consider using single-cell approaches or isolating specific cell populations.

What is the evidence linking FAM71F1 to human male infertility?

Emerging evidence connects FAM71F1 dysfunction to male infertility:

• Expression studies: FAM71F1 expression is significantly downregulated in patients with male infertility , suggesting its potential involvement in human reproductive pathologies.

• Functional insights from animal models: FAM71F1-mutant mice exhibit complete male infertility due to abnormal acrosome formation and impaired acrosome reaction . These phenotypes resemble certain forms of human globozoospermia (round-headed sperm syndrome).

• Molecular mechanism: The demonstrated role of FAM71F1 in regulating vesicle trafficking during acrosome biogenesis provides a plausible mechanistic link to human male infertility cases characterized by acrosomal defects.

Researchers investigating human male infertility should consider including FAM71F1 in candidate gene panels, particularly for cases presenting with acrosomal abnormalities or globozoospermia. Immunohistochemical analysis of testicular biopsies using FAM71F1 antibodies may provide valuable diagnostic information in selected cases.

What methodologies are recommended for investigating FAM71F1 in human fertility studies?

For human fertility research focusing on FAM71F1:

• Genetic screening: Sequence the FAM71F1 gene in infertile male patients, particularly those with acrosomal abnormalities. Focus on coding regions and regulatory elements.

• Expression analysis: Perform quantitative RT-PCR and immunohistochemistry on testicular biopsies from infertile patients versus fertile controls.

• Proteomic approaches: Use mass spectrometry to identify altered protein interactions in samples from infertile patients.

• Functional assays: Assess acrosome reaction in patient sperm samples using calcium ionophore challenge and FAM71F1 immunostaining.

• Cell models: Utilize patient-derived induced pluripotent stem cells differentiated toward the male germ cell lineage to study FAM71F1 function in a human context.

What are promising areas for future research on FAM71F1?

Several promising research directions for FAM71F1 include:

• Regulatory mechanisms: Investigate transcriptional and post-transcriptional regulation of FAM71F1 during spermatogenesis.

• Structural biology: Determine the three-dimensional structure of FAM71F1 and its complex with RAB2A/B to understand binding specificity and potential drug targeting.

• Broader physiological roles: Examine potential functions of FAM71F1 in tissues beyond the testis, particularly in cells with specialized vesicular trafficking requirements.

• Clinical applications: Develop diagnostic tests for FAM71F1-related male infertility and explore therapeutic approaches to restore proper acrosome formation.

• Comparative biology: Study FAM71F1 across species to understand evolutionary conservation and divergence of acrosome formation mechanisms.

These research directions will contribute to a more comprehensive understanding of FAM71F1's biological functions and potential clinical applications in reproductive medicine.