REC107 Antibody

What is REC107 and why is it important in meiotic recombination research?

REC107 (also known as MER2, YJR021C, or J1462) is a meiotic recombination protein in Saccharomyces cerevisiae that plays a critical role in the initiation of meiotic recombination. The protein is approximately 50 kDa in size and functions in the tethering of recombination initiation proteins . Research into REC107 is important for understanding fundamental mechanisms of genetic recombination during meiosis, which has implications for genome stability and evolution.

The methodological approach to studying REC107 typically involves using antibodies that recognize specific epitopes, such as the amino acid region 26-35, which allows researchers to track protein expression, localization, and modification during meiotic processes .

What applications are REC107 antibodies validated for in laboratory research?

REC107 antibodies have been validated for multiple research applications:

These applications enable researchers to study REC107's expression levels, post-translational modifications, and protein-protein interactions in various experimental contexts . For optimal results, researchers should validate the specific dilutions with their experimental systems, as sensitivity can vary based on sample preparation and detection methods.

How should researchers optimize Western blot protocols for REC107 detection?

For optimal Western blot detection of REC107 protein:

• Sample preparation: Extract proteins from yeast cells during meiotic stages when REC107 expression is highest

• Gel selection: Use 10-12% SDS-PAGE gels to effectively resolve the ~50 kDa REC107 protein

• Transfer conditions: Semi-dry transfer at 15V for 30-45 minutes or wet transfer at 30V overnight at 4°C

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody: Apply anti-REC107 antibody at 1:1,000 - 1:10,000 dilution in blocking buffer

• Detection: The antibody reacts with both phosphorylated and unphosphorylated REC107 at the S30 position

It's critical to include appropriate positive controls and optimize incubation times based on your specific experimental conditions. The antibody storage buffer (20 mM KH₂PO₄, 150 mM NaCl, pH 7.2 with 0.01% sodium azide) should be considered when designing blocking and washing buffers to minimize background .

How can REC107 antibodies be used to investigate phosphorylation-dependent protein interactions during meiosis?

REC107/MER2 phosphorylation, particularly at the S30 position, is a key regulatory step in meiotic recombination initiation. To investigate phosphorylation-dependent interactions:

• Use immunoprecipitation with anti-REC107 antibodies to pull down protein complexes under different phosphorylation conditions

• Employ phospho-specific antibodies in parallel with general REC107 antibodies to distinguish phosphorylated from unphosphorylated forms

• Implement phos-tag gel electrophoresis followed by Western blotting to separate phosphorylated species

• Design comparative experiments with phosphomimetic and phospho-deficient yeast mutants

The available REC107 antibodies react with both phosphorylated and unphosphorylated forms at the S30 position , enabling researchers to track total protein levels while using phospho-specific antibodies to monitor specific modification states.

For complex interaction studies, consider:

What are the critical considerations when comparing different commercial REC107 antibodies for reproducibility in research?

When comparing different commercial antibodies against REC107:

• Epitope specificity: Verify the exact epitope region recognized. The antibodies documented (amino acids 26-35 of S. cerevisiae MER2) target a critical region that may affect function detection

• Validation methods: Assess how thoroughly the antibody has been validated:

  • Knockout/knockdown controls

  • Peptide competition assays

  • Cross-reactivity testing with related proteins

• Production consistency: Polyclonal antibodies may show lot-to-lot variation. Consider:

• Application-specific validation: The documented antibodies are validated for ELISA (1:5,000-1:25,000), Western blot (1:1,000-1:10,000), and immunoprecipitation (1:100) . Verify performance in your specific application.

• Cross-species reactivity: Current antibodies are specifically reactive to Saccharomyces cerevisiae . For comparative studies across species, additional validation is necessary.

How might researchers design experiments to investigate REC107's role in meiotic DNA double-strand break formation?

To investigate REC107's role in meiotic DNA double-strand break (DSB) formation:

• Temporal analysis protocol:

  • Synchronize yeast cultures entering meiosis

  • Collect samples at defined time points (0, 1, 2, 3, 4, 5, 6 hours after induction)

  • Process for both protein analysis (Western blot with anti-REC107) and DNA break analysis (pulsed-field gel electrophoresis)

  • Correlate REC107 phosphorylation state with DSB timing

• Genetic interaction mapping:

  • Create a panel of strains with mutations in REC107 and known DSB machinery components

  • Use anti-REC107 antibodies to assess protein expression, localization, and modification

  • Quantify DSB formation using Southern blotting at hotspots

  • Establish epistasis relationships based on phenotypic outcomes

• Chromatin association dynamics:

  • Perform chromatin immunoprecipitation (ChIP) using anti-REC107 antibodies

  • Analyze temporal association with recombination hotspots

  • Compare wild-type versus mutant backgrounds

What are the optimal storage and handling conditions for maintaining REC107 antibody activity?

For optimal maintenance of REC107 antibody activity:

• Short-term storage (up to 1 month):

  • Store at 4°C in the provided buffer (20 mM KH₂PO₄, 150 mM NaCl, pH 7.2 with 0.01% sodium azide)

  • Avoid repeated freeze-thaw cycles

• Long-term storage:

  • Store at -20°C

  • Aliquot into single-use volumes to avoid repeated freeze-thaw cycles

  • Typical antibody concentration is 0.75 mg/mL

• Working solution preparation:

  • Dilute only the required amount in appropriate buffer

  • For Western blotting, prepare fresh working solutions

  • Include stabilizing proteins (BSA, gelatin) for very dilute working solutions

• Contamination prevention:

  • Use sterile technique when handling antibodies

  • The commercial products are sterile filtered

  • Sodium azide (0.01%) is included as a preservative but is incompatible with HRP-based detection systems

• Stability monitoring:

  • Include positive controls in each experiment to verify continued activity

  • Document lot numbers and monitor for consistency

How can researchers troubleshoot non-specific binding issues with REC107 antibodies?

When encountering non-specific binding with REC107 antibodies:

• Optimize blocking conditions:

  • Test different blocking agents (5% milk, 3-5% BSA, commercial blockers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Include 0.1-0.3% Tween-20 in washing buffers

• Adjust antibody conditions:

  • Titrate antibody concentration (start with recommended 1:1,000 - 1:10,000 for Western blot)

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

  • Pre-adsorb antibody with yeast lysate from REC107-knockout strain

• Employ additional controls:

  • Include REC107-knockout/knockdown samples as negative controls

  • Use competing peptide (amino acids 26-35 of REC107) to confirm specificity

  • Include gradient of purified recombinant REC107 protein

• Sample preparation refinements:

  • Optimize lysis conditions to reduce background proteins

  • Consider subcellular fractionation to enrich for nuclear proteins

  • Implement additional purification steps before immunodetection

What considerations should be made when designing immunofluorescence experiments with REC107 antibodies?

When designing immunofluorescence experiments using REC107 antibodies:

• Fixation optimization:

  • Test multiple fixation methods (4% paraformaldehyde, methanol/acetone)

  • Optimize fixation time (10-20 minutes) to preserve epitope accessibility

  • Consider dual fixation protocols for challenging epitopes

• Permeabilization considerations:

  • Yeast cell wall requires special attention - use zymolyase or lyticase treatment

  • Titrate permeabilization reagent (0.1-0.5% Triton X-100 or 0.05-0.2% SDS)

  • Balance permeabilization with epitope preservation

• Antibody validation strategy:

  • Begin with Western blot confirmation before attempting immunofluorescence

  • Include REC107 knockout/knockdown controls

  • Consider epitope-tagged REC107 constructs as positive controls

• Signal amplification:

  • Primary antibody concentration: start at 1:100-1:500 dilution

  • Extended incubation (overnight at 4°C)

  • Consider tyramide signal amplification for low abundance targets

• Co-localization experiments:

  • Combine with established meiotic markers (e.g., Rad51, synaptonemal complex proteins)

  • Use appropriate filter sets to minimize bleed-through

  • Include single-label controls

Since the documented REC107 antibodies do not explicitly list immunofluorescence as a validated application , researchers should conduct preliminary validation studies before proceeding with extensive experiments.

How can mass spectrometry be integrated with REC107 immunoprecipitation to identify novel interaction partners?

To integrate mass spectrometry (MS) with REC107 immunoprecipitation for interaction partner discovery:

• Optimized immunoprecipitation protocol:

  • Scale up cell culture (1-2 liters of yeast culture at appropriate meiotic timepoints)

  • Use mild lysis conditions to preserve protein-protein interactions

  • Implement the validated 1:100 dilution of anti-REC107 antibody

  • Include appropriate negative controls (IgG, pre-immune serum)

• Sample preparation for MS:

  • On-bead digestion with high-quality trypsin

  • Peptide clean-up using stage tips or similar technology

  • Consider chemical crosslinking prior to lysis for transient interactions

• MS analytical strategy:

  • Employ high-resolution MS (Orbitrap or similar)

  • Use quantitative approaches (SILAC, TMT, label-free quantification)

  • Focus on proteins enriched compared to control samples

• Interaction validation pipeline:

  • Confirm key interactions by reciprocal immunoprecipitation

  • Perform directed yeast two-hybrid or proximity ligation assays

  • Generate interaction network maps with confidence scores

What strategies can be employed to study temporal dynamics of REC107 phosphorylation during meiosis?

To study the temporal dynamics of REC107 phosphorylation during meiosis:

• Synchronization and sampling approach:

  • Implement established yeast meiotic synchronization protocols

  • Collect samples at short intervals (15-30 minutes) throughout meiotic progression

  • Process samples identically for consistent comparison

• Phosphorylation detection methods:

  • Phos-tag SDS-PAGE followed by Western blotting with anti-REC107 antibody

  • Parallel blotting with phospho-specific antibodies (if available)

  • Mass spectrometry-based phosphopeptide enrichment and quantification

• Kinetics analysis techniques:

  • Quantify band intensity ratios of phosphorylated vs. non-phosphorylated forms

  • Plot temporal profiles of different phosphorylation states

  • Correlate with meiotic markers and cellular events

• Genetic perturbation experiments:

  • Analyze phosphorylation patterns in kinase/phosphatase mutants

  • Create phosphomimetic (S→D/E) and phospho-deficient (S→A) mutants of key sites

  • Monitor functional consequences in parallel with phosphorylation status

Since the available REC107 antibodies react with both phosphorylated and unphosphorylated forms at the S30 position , researchers can track total protein while using mobility shifts or phospho-specific antibodies to monitor modification state changes.

How can computational approaches complement experimental REC107 antibody data for structural and functional insights?

Computational approaches can significantly enhance experimental REC107 antibody data:

• Epitope analysis and antibody binding prediction:

  • Map the recognized epitope (amino acids 26-35) on predicted protein structures

  • Assess epitope accessibility in different protein conformations

  • Predict potential post-translational modifications that might affect antibody binding

• Structural modeling integration:

  • Generate homology models of REC107/MER2 protein

  • Map experimentally identified phosphorylation sites onto the model

  • Simulate structural changes upon phosphorylation

  • Predict protein-protein interaction interfaces

• Systems biology approaches:

  • Integrate antibody-derived protein expression/modification data into meiotic regulatory networks

  • Apply machine learning to predict functional partners based on co-expression patterns

  • Use active learning algorithms to optimize experimental design for validation studies

• Advanced imaging analysis:

  • Apply deconvolution algorithms to immunofluorescence images

  • Implement 3D reconstruction of protein localization

  • Quantify co-localization coefficients with known meiotic markers

Recent advances in antibody-antigen binding prediction can be leveraged to optimize experimental conditions. The library-on-library approaches and active learning strategies described for antibody-antigen binding prediction could be applied to refine REC107 antibody applications .

How might CRISPR-Cas9 genome editing be combined with REC107 antibody-based approaches to study meiotic recombination mechanisms?

Integrating CRISPR-Cas9 genome editing with REC107 antibody-based approaches:

• Precision engineering of REC107/MER2:

  • Generate epitope-tagged versions at endogenous loci

  • Create specific point mutations at key phosphorylation sites

  • Develop conditional knockout/knockdown systems

  • Design fluorescent protein fusions for live imaging

• Combined analytical workflow:

  • Validate edited strains using anti-REC107 antibodies via Western blotting

  • Compare protein expression/modification levels between wildtype and edited strains

  • Assess functional consequences using established meiotic phenotyping

  • Map protein-protein interactions using antibody-based approaches

• Multiplexed genetic analysis:

  • Generate combinatorial mutations in REC107 and interacting partners

  • Apply antibody-based detection to assess epistatic relationships

  • Create comprehensive interaction networks through systematic analysis

• Spatiotemporal regulation studies:

  • Implement optogenetic or chemically inducible systems to control REC107 function

  • Use antibodies to verify protein presence/absence after induction

  • Monitor consequent effects on downstream processes

This integrated approach allows researchers to connect genetic perturbations directly to protein-level consequences, providing mechanistic insights into REC107's functions during meiotic recombination.

What considerations are important when designing ChIP-seq experiments using REC107 antibodies?

When designing Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) with REC107 antibodies:

• Antibody validation for ChIP applications:

  • Perform preliminary ChIP-qPCR at known binding sites

  • Assess enrichment over input and IgG controls

  • Verify specificity using knockout/knockdown controls

  • Test multiple antibody concentrations to optimize signal-to-noise ratio

• Experimental design considerations:

  • Sample synchronized meiotic populations at key timepoints

  • Include appropriate controls (input, IgG, non-meiotic cells)

  • Consider parallel ChIP for known interacting proteins

  • Implement spike-in normalization for quantitative comparisons

• Protocol optimization for yeast ChIP-seq:

  • Crosslinking conditions (1% formaldehyde, 10-15 minutes)

  • Sonication parameters to achieve 200-500 bp fragments

  • Immunoprecipitation conditions (antibody amount, incubation time)

  • Washing stringency to minimize background

• Data analysis pipeline:

  • Align reads to appropriate yeast genome assembly

  • Call peaks using MACS2 or similar algorithms

  • Perform differential binding analysis between conditions

  • Integrate with other genomic datasets (RNA-seq, Hi-C)

While ChIP is not explicitly listed as a validated application for the documented REC107 antibodies , researchers experienced with ChIP optimization may be able to adapt these antibodies for chromatin studies.

How can single-cell approaches be combined with REC107 antibody detection to study meiotic heterogeneity?

Combining single-cell approaches with REC107 antibody detection:

• Single-cell immunofluorescence strategies:

  • Optimize fixation and permeabilization for single yeast cells

  • Implement high-content imaging systems for automated analysis

  • Develop quantitative image analysis pipelines for protein levels and localization

  • Correlate with cell cycle/meiotic stage markers

• Mass cytometry (CyTOF) approaches:

  • Conjugate anti-REC107 antibodies with rare earth metals

  • Develop panels including multiple meiotic proteins and modifications

  • Implement dimensionality reduction techniques for population analysis

  • Identify distinct meiotic substates based on protein expression patterns

• Single-cell Western blotting:

  • Adapt microfluidic systems for yeast cell isolation

  • Optimize lysis conditions for single-cell protein extraction

  • Scale antibody dilutions appropriately for reduced protein amounts

  • Develop sensitive detection methods for low-abundance proteins

• Integration with single-cell genomics:

  • Combine protein detection with DNA damage mapping techniques

  • Correlate REC107 levels/modifications with recombination outcomes

  • Develop computational approaches to link protein states to genomic events