REC114 Antibody

What is REC114 and why is it significant in meiotic research?

REC114 is an evolutionarily conserved protein essential for programmed meiotic DNA double-strand break (DSB) formation. It works in conjunction with other proteins like MEI4 and IHO1 to facilitate the homologous recombination pathway during meiosis. This pathway is critical for proper chromosome segregation at the first meiotic division . Mouse REC114 has been demonstrated to be required for meiotic DSB formation, similar to its orthologs in other organisms. It forms a complex with MEI4 and IHO1 on the axes of meiotic chromosomes, making it a crucial target for researchers studying meiotic processes and fertility .

What types of REC114 antibodies are available for research?

Based on published research protocols, chicken anti-REC114 antibodies have been successfully generated against mouse REC114 protein and affinity-purified for experimental use . These antibodies have been validated in several experimental contexts including immunoprecipitation, Western blot analysis, and immunofluorescence studies. Researchers should note that antibody selection should consider the species specificity, clonality (monoclonal vs polyclonal), and the application intended (Western blot, immunoprecipitation, immunofluorescence, etc.) .

How do I validate a REC114 antibody for my experiments?

Validation of REC114 antibodies should follow these methodological steps:

• Knockout/Negative Control Testing: Compare antibody reactivity in wild-type samples versus Rec114-/- tissues or cells. Published research shows that proper antibodies should show no specific signal in knockout samples .

• Western Blot Analysis: Verify that the antibody detects a protein of the expected molecular weight in whole testis protein extracts. The antibody should show significantly reduced or absent signal in Rec114-/- samples .

• Immunoprecipitation Efficiency: Test the antibody's ability to specifically pull down REC114 and its known interaction partners (MEI4 and IHO1) .

• Immunofluorescence Pattern: In cytological preparations, REC114 antibodies should detect foci that colocalize with MEI4 on chromosome axes during early meiotic prophase, with numbers highest at leptonema and progressively decreasing upon synapsis .

How can REC114 antibodies be used to study protein-protein interactions in meiosis?

REC114 antibodies serve as powerful tools for investigating meiotic protein complexes through several advanced approaches:

• Co-immunoprecipitation (Co-IP): REC114 antibodies have been effectively used to pull down REC114 and identify its interaction partners. Research has shown that REC114 immunoprecipitation can co-precipitate MEI4 and IHO1, suggesting these proteins form a complex in vivo . The protocol involves:

  • Preparing whole testis protein extracts

  • Incubating with affinity-purified chicken anti-REC114 antibody

  • Capturing complexes with agarose-immobilized anti-chicken IgY Fc

  • Analyzing associated proteins via Western blot

• Reverse Co-IP: Researchers can perform the reverse experiment using MEI4 antibodies to confirm the interaction. Studies have shown that MEI4 immunoprecipitation can pull down both REC114 and IHO1, further supporting their association in a complex .

• Interaction Domain Mapping: Combined with molecular approaches like Y2H (yeast two-hybrid) assays and recombinant protein expression, REC114 antibodies can help validate interactions between specific domains, such as the demonstrated interaction between REC114 C-terminal domain and MEI4 N-terminal domain .

What methodological approaches can resolve contradictory REC114 localization data across different studies?

When facing conflicting REC114 localization data, researchers should implement the following methodological approaches:

• Fixation Protocol Comparison: Systematically test different fixation methods (paraformaldehyde, methanol-acetone, etc.) as epitope accessibility can vary dramatically with fixation conditions.

• Multiple Antibody Validation: Use antibodies raised against different regions of REC114 to ensure consistent localization patterns and rule out epitope masking during certain meiotic stages.

• Sequential Immunolabeling: Perform sequential labeling with REC114 antibody followed by antibodies against known interactors (MEI4, IHO1) to precisely determine colocalization patterns. Research has shown that REC114 foci colocalize with MEI4 and their numbers are highest at leptonema before progressively decreasing upon synapsis .

• Genetic Knockout Controls: Include parallel experiments in Rec114-/-, Mei4-/-, and Spo11-/- samples to distinguish specific from non-specific signals. Studies have demonstrated that in Mei4-/- spermatocytes, REC114 foci are reduced by more than 10-fold compared to wild type cells .

• Super-resolution Microscopy: When conventional microscopy yields contradictory results, super-resolution techniques can provide higher precision in localizing REC114 relative to chromosome axes.

How can REC114 antibodies help investigate the ANKRD31-REC114 interaction in meiotic DSB formation?

The ANKRD31-REC114 interaction has emerged as crucial for controlling DSB locations, timing, and number . Researchers can employ REC114 antibodies to:

• Map Interaction Domains: Combined with protein interaction assays, REC114 antibodies can help validate which domains of REC114 (particularly the N-terminal domain) interact with ANKRD31 .

• Examine Colocalization Patterns: Using immunofluorescence with REC114 and ANKRD31 antibodies, researchers can assess whether mutations in ANKRD31 (such as ANKRD31-EA and ANKRD31-ΔC) affect REC114 localization patterns .

• Quantify Interaction Strength: Immunoprecipitation with REC114 antibodies followed by Western blotting for ANKRD31 can help quantify how specific mutations affect the stability of this interaction in vivo .

• Assess Functional Outcomes: Combining REC114 antibody staining with markers of DSB formation (γH2AX) and repair (RAD51, DMC1) allows researchers to correlate interaction defects with phenotypic outcomes in mutant backgrounds .

What are the optimal protocols for immunoprecipitation using REC114 antibodies?

Based on published research, the following protocol has been validated for REC114 immunoprecipitation:

REC114 Immunoprecipitation Protocol:

• Sample Preparation:

  • Prepare whole testis protein extracts according to established protocols

  • Use approximately 1.5mg of extract diluted in IP buffer (20mM Tris-HCl; 150mM NaCl; 0.05% NP40; 0.1% Tween-20; 10% glycerol; protease inhibitors)

• Antibody Incubation:

  • Add 2 μg of affinity-purified chicken anti-REC114 antibody

  • Incubate at 4°C overnight with gentle rotation

• Bead Capture:

  • Add 50 μl of agarose-immobilized anti-chicken IgY Fc (goat)

  • Incubate at 4°C for 1 hour with gentle rotation

• Washing:

  • Wash beads five times with washing buffer (20mM Tris-HCl pH 7.5, 0.05% NP-40, 0.1% Tween-20, 10% glycerol, 150mM NaCl)

• Elution:

  • Elute immunoprecipitated material with 2X Laemmli loading buffer (containing 20mM DTT)

  • Incubate at 95°C for 5 minutes

• Analysis:

  • Analyze by SDS-PAGE followed by Western blotting

  • Use appropriate primary antibodies to detect REC114 and potential interacting partners

What controls are essential for REC114 immunofluorescence experiments?

For reliable REC114 immunofluorescence studies, the following controls are essential:

• Genetic Knockouts: Include Rec114-/- samples as negative controls to establish background staining levels .

• Comparative Controls: Include parallel staining of Mei4-/- and Spo11-/- samples to understand the dependency relationships between these proteins. Research has shown that REC114 foci are drastically reduced in Mei4-/- spermatocytes but not in Spo11-/- mice .

• Secondary Antibody Controls: Include samples stained with only secondary antibody to evaluate non-specific binding.

• Co-staining Controls: Always co-stain with axis markers (SYCP3) and other meiotic stage-specific markers (SYCP1 for synapsis) to accurately identify meiotic substages .

• Blocking Peptide Controls: When available, include controls where the antibody is pre-incubated with the immunizing peptide to demonstrate staining specificity.

What is the recommended Western blot protocol for REC114 detection?

Based on published methodologies, the following Western blot protocol is recommended for REC114 detection:

Table 1: Optimized Western Blot Protocol for REC114 Detection

For optimal results, researchers should include SYCP3 detection (1/3000 dilution) as a loading control for testis extracts .

How can researchers address weak or absent REC114 signals in immunofluorescence experiments?

When facing weak or absent REC114 signals in immunofluorescence experiments, consider these methodological solutions:

• Epitope Retrieval Optimization: Try different antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Enzymatic retrieval with proteinase K

  • Extended permeabilization with higher concentrations of Triton X-100

• Signal Amplification: Implement signal enhancement techniques:

  • Use tyramide signal amplification (TSA)

  • Try biotin-streptavidin amplification systems

  • Consider higher sensitivity detection systems

• Antibody Concentration Titration: Systematically test a range of primary antibody concentrations (1:100 to 1:2000) to identify the optimal signal-to-noise ratio.

• Sample Preparation Stage: REC114 focus numbers are highest at leptonema and progressively decrease upon synapsis. Ensure you're examining the appropriate meiotic stage .

• Mutual Dependency Consideration: Remember that REC114 and MEI4 are reciprocally required for their localization. If working with Mei4-/- samples, expect a significant reduction in REC114 foci .

What strategies can resolve non-specific bands in REC114 Western blots?

When troubleshooting non-specific bands in REC114 Western blots, implement these targeted approaches:

• Adjust Reducing Conditions: Published protocols show that adding 10mM DTT specifically to samples for REC114 detection can improve specificity .

• Modify Antibody Incubation Parameters:

  • Try lower antibody concentrations

  • Reduce incubation temperature (4°C instead of room temperature)

  • Add 0.1-0.5% non-ionic detergent to reduce non-specific binding

• Use Genetic Controls: Always include Rec114-/- samples as negative controls to distinguish specific from non-specific bands .

• Try Alternative Blocking Agents:

  • Replace milk with 5% BSA if phosphorylated proteins are being detected

  • Consider commercial blocking reagents optimized for chicken antibodies

• Secondary Antibody Selection: Use highly cross-adsorbed secondary antibodies specifically designed for chicken primary antibodies, such as donkey anti-chicken IgY (1/3000) .

How might REC114 antibodies contribute to understanding the structural biology of meiotic DSB machinery?

REC114 antibodies can be instrumental for advancing structural biology insights through:

• Immuno-Electron Microscopy: Using REC114 antibodies conjugated to gold particles could reveal the ultrastructural organization of REC114 complexes on meiotic chromosome axes.

• Chromatin Immunoprecipitation Sequencing (ChIP-seq): REC114 antibodies could help map genome-wide binding sites, potentially revealing sequence preferences or chromatin features that promote DSB formation.

• In situ Proximity Ligation Assays (PLA): Combining REC114 antibodies with antibodies against other DSB proteins in PLA experiments could visually map the spatial relationships within the DSB machinery complex.

• Co-IP Coupled to Mass Spectrometry: Using REC114 antibodies for immunoprecipitation followed by mass spectrometry could identify novel interaction partners beyond the known MEI4 and IHO1 associations .

• Cryo-EM Studies: REC114 antibodies could be used to validate cryo-EM structural models, particularly focusing on how the REC114 N-terminal Pleckstrin Homology domain and its dimerization properties contribute to DSB machinery architecture .

How can REC114 antibodies help investigate the relationship between ANKRD31-REC114 interaction and fertility disorders?

REC114 antibodies provide several methodological approaches to connect basic research on ANKRD31-REC114 interactions to fertility disorders:

• Patient Sample Analysis: REC114 antibodies could be used to examine REC114 localization patterns in testicular biopsies from infertility patients, potentially identifying cases where improper REC114 localization correlates with meiotic defects.

• Model Organism Studies: By combining REC114 antibody staining with genetic models carrying mutations in ANKRD31 (such as ANKRD31-EA), researchers can quantitatively assess how interaction defects correlate with reduced DSB formation, abnormal recombination, and fertility outcomes .

• High-throughput Screening: REC114 antibodies could be adapted for high-content imaging to screen for compounds that enhance or restore defective ANKRD31-REC114 interactions in cellular models.

• Biomarker Development: Quantitative assessment of REC114-ANKRD31 interaction using proximity-based assays with REC114 antibodies could potentially serve as a biomarker for certain forms of male infertility.