SPAC4G9.19 Antibody

What is SPAC4G9.19 and what cellular functions does it have in fission yeast?

SPAC4G9.19 (UniProt No. Q10247) is a protein found in Schizosaccharomyces pombe (fission yeast, strain 972 / ATCC 24843). While the specific function of this protein requires further investigation, it's studied in the context of fission yeast cellular processes. Current research suggests its involvement in cellular pathways similar to those targeted in antibody research for other model organisms. Methodologically, researchers can investigate its function through knockout studies, localization experiments using the SPAC4G9.19 Antibody, and interaction studies with known pathway components in S. pombe .

What are the optimal storage conditions for maintaining SPAC4G9.19 Antibody activity?

For maximum stability and activity retention, SPAC4G9.19 Antibody should be stored at either -20°C or -80°C upon receipt. Critically important is avoiding repeated freeze-thaw cycles, as these can significantly reduce antibody functionality through protein denaturation and aggregation. The antibody is provided in a protective storage buffer containing 0.03% Proclin 300 as a preservative and 50% Glycerol in 0.01M PBS (pH 7.4) to maintain stability during storage . For daily experimental work, small aliquots should be prepared to minimize freeze-thaw damage.

What validation methods confirm SPAC4G9.19 Antibody specificity?

SPAC4G9.19 Antibody has been validated through ELISA and Western Blot (WB) applications to ensure proper identification of the target antigen. As a polyclonal antibody raised against recombinant Schizosaccharomyces pombe SPAC4G9.19 protein, it undergoes antigen affinity purification to enhance specificity. For researchers conducting independent validation, recommended methods include:

• Positive control: Using recombinant SPAC4G9.19 protein

• Negative control: Testing against lysates from organisms other than S. pombe

• Knockout validation: Testing against SPAC4G9.19 knockout strains when available

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide

How can researchers optimize Western blot protocols when using SPAC4G9.19 Antibody?

Western blot optimization for SPAC4G9.19 Antibody requires careful attention to several parameters:

When working with fission yeast samples, cell lysis requires optimization due to the rigid cell wall. Using glass bead disruption in combination with a buffer containing protease inhibitors is recommended to preserve protein integrity. Include positive and negative controls in each experiment, and validate bands using molecular weight markers corresponding to the expected size of SPAC4G9.19 .

What considerations are important when designing colocalization studies using SPAC4G9.19 Antibody?

Colocalization studies require careful planning when using SPAC4G9.19 Antibody:

• Fixation method selection: Aldehyde-based fixatives may preserve antigenicity better than organic solvents for this particular antibody. Perform pilot experiments comparing 4% paraformaldehyde with methanol fixation.

• Permeabilization optimization: S. pombe cell wall requires specialized permeabilization. A suggested approach is enzymatic digestion with zymolyase (0.5-1 mg/ml) followed by 0.1% Triton X-100 treatment.

• Antibody combinations: Since SPAC4G9.19 Antibody is raised in rabbit, pair with mouse-derived antibodies against other cellular markers for dual labeling.

• Controls for cross-reactivity: Include single-antibody controls to ensure secondary antibodies do not cross-react.

• Quantitative analysis: Use colocalization coefficients (Pearson's, Manders') rather than visual assessment alone. Establish thresholds based on negative controls .

How does the polyclonal nature of SPAC4G9.19 Antibody influence experimental design and data interpretation?

The polyclonal nature of SPAC4G9.19 Antibody has several significant implications for research design:

SPAC4G9.19 Antibody is developed by immunizing rabbits with recombinant S. pombe SPAC4G9.19 protein, resulting in antibodies that recognize multiple epitopes on the target protein. This characteristic influences experimental approaches in several ways:

What strategies can enhance SPAC4G9.19 Antibody specificity in challenging applications?

Enhancing specificity is crucial when working with complex samples or in sensitive applications:

• Sample preparation refinement:

  • For S. pombe lysates, subcellular fractionation can reduce complexity

  • Enrichment of relevant compartments may improve signal-to-noise ratio

  • Consider native versus denaturing conditions based on epitope accessibility

• Pre-absorption protocol:

  • Incubate antibody with non-target proteins from same or similar organisms

  • Use 5-10 μg of control lysate per 1 μg antibody (4°C, 1-2 hours)

  • Centrifuge at 14,000g for 10 minutes to remove complexes before use

• Alternative validation approaches:

  • Employ orthogonal techniques (mass spectrometry, RNA analysis)

  • Compare localizations with tagged versions of SPAC4G9.19

  • Consider parallel experiments with CRISPR-modified strains

• Buffer and condition optimization:

  • Test different pH ranges (6.8-8.0) in binding buffers

  • Adjust salt concentrations (150-500 mM NaCl) to reduce non-specific interactions

  • Evaluate detergent types and concentrations for membrane-associated studies

How can SPAC4G9.19 Antibody be utilized in studying cell cycle regulation in fission yeast?

SPAC4G9.19 Antibody can be strategically employed to investigate potential roles in cell cycle regulation:

• Synchronization protocols:

  • Implement nitrogen starvation or lactose gradient centrifugation for cell synchronization

  • Collect samples at 20-minute intervals across the cell cycle

  • Process for Western blot or immunofluorescence with SPAC4G9.19 Antibody

  • Quantify protein levels and correlate with cell cycle markers

• Co-immunoprecipitation approach:

  • Lyse synchronized cells in buffer containing 1% NP-40, 150 mM NaCl, 50 mM Tris pH 7.5

  • Pre-clear lysate with Protein A/G beads

  • Immunoprecipitate with SPAC4G9.19 Antibody overnight

  • Analyze precipitates for known cell cycle regulators

• Chromatin association studies:

  • Fractionate cells into cytoplasmic, nuclear soluble, and chromatin-bound fractions

  • Analyze SPAC4G9.19 distribution across fractions during cell cycle progression

  • Correlate with DNA replication timing and chromosome condensation

What considerations are important when using SPAC4G9.19 Antibody for quantitative applications?

Quantitative applications require rigorous standardization:

• Standard curve development:

  • Use purified recombinant SPAC4G9.19 protein in known quantities

  • Create 5-point standard curves with 2-fold dilutions

  • Process standards alongside experimental samples

• Normalization strategy:

  • Select appropriate loading controls stable under your experimental conditions

  • Consider dual normalization (total protein + housekeeping protein)

  • Validate normalization method stability across your experimental conditions

• Imaging and quantification parameters:

  • For fluorescence applications, establish linear dynamic range

  • Use sub-saturating exposure times

  • Apply consistent analysis parameters across all samples

  • Implement blind analysis when possible to reduce bias

• Technical replication:

  • Minimum of three technical replicates per biological sample

  • Address outliers using statistically sound methods

  • Report variability measures alongside means

How does SPAC4G9.19 Antibody compare methodologically with other tools for studying this protein?

When designing comprehensive research strategies, consider these comparative advantages:

For optimal experimental design, researchers often implement parallel approaches, using SPAC4G9.19 Antibody alongside complementary techniques to build comprehensive understanding of protein function in fission yeast biology .

What approaches can resolve inconsistent results when using SPAC4G9.19 Antibody across different experimental setups?

Inconsistent results with SPAC4G9.19 Antibody may stem from several factors:

• Epitope masking investigation:

  • Test alternative sample preparation methods (native vs. denatured)

  • Evaluate different detergent types (SDS, NP-40, Triton X-100) for extraction

  • Consider epitope retrieval methods for fixed samples

• Antibody handling evaluation:

  • Review storage conditions and freeze-thaw history

  • Test freshly reconstituted aliquots

  • Validate antibody performance with positive controls

• Protocol standardization:

  • Document all protocol steps in precise detail

  • Control incubation temperatures rigorously

  • Standardize reagent preparation methods

• Sample variability assessment:

  • Ensure consistent growth conditions for S. pombe cultures

  • Standardize cell density and growth phase

  • Account for stress responses that may alter protein expression

How can researchers validate contradictory findings between SPAC4G9.19 Antibody results and other experimental approaches?

When faced with contradictory results:

• Methodological triangulation:

  • Implement at least three independent methodologies

  • Consider both antibody-dependent and antibody-independent approaches

  • Evaluate the limitations inherent to each method

• Biological context examination:

  • Test under varied growth conditions, stressors, or genetic backgrounds

  • Consider cell cycle, nutritional status, or population heterogeneity

  • Evaluate temporal dynamics that might explain discrepancies

• Technical validation approach:

  • Exchange materials between laboratories if collaborative

  • Blind sample analysis to reduce confirmation bias

  • Sequence verify strains and plasmids being used

• Statistical rigor enhancement:

  • Increase biological replication

  • Apply appropriate statistical tests for data distribution

  • Consider Bayesian approaches for integrating conflicting data sets

How might SPAC4G9.19 Antibody applications evolve with advances in super-resolution microscopy?

As super-resolution techniques develop, SPAC4G9.19 Antibody applications will expand:

• Nanoscale localization studies:

  • Implementation in STORM/PALM microscopy requires secondary antibodies conjugated with photo-switchable fluorophores

  • Combination with structured illumination microscopy (SIM) can reveal previously undetectable protein distributions

  • Protocol modifications needed include decreased antibody concentrations and enhanced blocking

• Multi-protein complex visualization:

  • Expansion microscopy compatibility allows physical separation of crowded epitopes

  • DNA-PAINT techniques can enable multiplexed detection with other fission yeast proteins

  • Correlative light-electron microscopy approaches can connect protein localization with ultrastructure

• Quantitative single-molecule approaches:

  • Single-molecule pull-down (SiMPull) techniques can determine absolute stoichiometry

  • Direct stochastic optical reconstruction microscopy (dSTORM) can quantify cluster sizes and distributions

  • These applications require rigorous antibody validation for single-molecule sensitivity

What emerging technologies might enhance the research value of SPAC4G9.19 Antibody?

Several emerging technologies show particular promise:

• Proximity labeling integration:

  • Combining antibody-based purification with BioID or APEX2 proximity labeling

  • Creating antibody-enzyme fusions for direct application in proximity studies

  • Enabling temporally controlled interaction mapping with inducible systems

• Microfluidic applications:

  • Single-cell antibody-based assays in droplet systems

  • Continuous monitoring of protein dynamics in microfluidic yeast cultures

  • High-throughput screening of genetic interactions affecting SPAC4G9.19

• Computational prediction integration:

  • Using antibody-validated localizations to refine AI-based protein interaction predictions

  • Combining experimental antibody data with in silico structural models

  • Developing machine learning approaches to predict epitopes for improved antibody design