SPAC22A12.08c Antibody

What is SPAC22A12.08c and why is it significant in Schizosaccharomyces pombe research?

SPAC22A12.08c is a gene/protein in Schizosaccharomyces pombe (fission yeast) identified with UniProt accession number O13899. While specific function characterization is ongoing, it belongs to a group of proteins found in the widely-used S. pombe strain 972 / ATCC 24843 . Researchers study this protein to understand fundamental cellular processes in fission yeast, which serves as an important model organism for eukaryotic cell biology, particularly for cell cycle regulation and division mechanisms.

The significance of studying SPAC22A12.08c stems from S. pombe's position as one of the simplest eukaryotic genomes (~5100 genes) . As demonstrated in genome reduction studies, understanding individual protein functions contributes to identifying minimal gene sets required for cellular viability under laboratory conditions.

What applications are most suitable for SPAC22A12.08c antibodies in fission yeast research?

SPAC22A12.08c antibodies can be employed in multiple experimental approaches:

Methanol fixation protocols are particularly effective for S. pombe proteins as outlined in studies of cell wall proteins .

How should researchers validate the specificity of SPAC22A12.08c antibody in experimental designs?

Antibody validation requires multiple approaches to ensure specificity:

• Genetic validation: Use a SPAC22A12.08c deletion strain (if viable) or a conditional knockdown strain as a negative control. Genome reduction studies in S. pombe provide protocols for gene deletion that could be adapted .

• Protein tagging comparison: Express epitope-tagged SPAC22A12.08c (e.g., HA, FLAG, or GFP tags) and compare localization/expression patterns between the tagged protein detection and antibody detection.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein. Similar approaches were used to validate antibody specificity in studies of S. pombe Sup11p .

• Preabsorption control: Incubate the antibody with purified recombinant SPAC22A12.08c protein before immunostaining to block specific binding sites.

• Cross-species reactivity assessment: Test the antibody against related proteins in other yeast species to evaluate potential cross-reactivity.

What are the optimal protein extraction methods when using SPAC22A12.08c antibody for Western blotting?

For effective protein extraction from fission yeast for SPAC22A12.08c detection:

• Cell wall disruption: Use glass bead lysis in buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 5 mM EDTA

  • 10% glycerol

  • 1% Triton X-100

  • Protease inhibitor cocktail

• Protease protection: Include both serine and cysteine protease inhibitors to prevent degradation during extraction .

How should researchers interpret contradictory localization data when using SPAC22A12.08c antibody?

When faced with contradictory localization data:

• Compare fixation methods: Different fixation methods can significantly affect epitope accessibility. Methanol fixation often preserves different epitopes than paraformaldehyde fixation in S. pombe .

• Evaluate antibody specificity under different conditions: Perform immunogold electron microscopy to obtain higher resolution localization data, as used in fission yeast cell wall protein studies .

• Cross-validate with tagged proteins: Express fluorescently-tagged SPAC22A12.08c and compare localization with antibody staining.

• Cell cycle dependence: Determine if localization varies throughout the cell cycle by synchronizing cultures using methods such as lactose gradient centrifugation or nitrogen starvation release.

• Stress response: Test if protein localization changes under different stress conditions (oxidative, temperature, nutrient limitation).

What approaches can resolve issues with high background in immunostaining experiments with SPAC22A12.08c antibody?

To reduce background and improve signal-to-noise ratio:

• Blocking optimization: Test different blocking agents:

  • 5% non-fat dry milk

  • 3-5% BSA

  • Commercial blocking reagents optimized for yeast samples

• Detergent screening: Test multiple detergents in wash buffers:

  Detergent	Concentration	Best For
  Triton X-100	0.1-0.3%	Membrane proteins
  Tween-20	0.05-0.1%	General applications
  SDS	0.01-0.1%	Stringent washing
  Saponin	0.1-0.5%	Preserving membrane structures

• Secondary antibody controls: Include samples with secondary antibody only to identify non-specific binding.

How can researchers use SPAC22A12.08c antibody to investigate protein-protein interactions in fission yeast?

For comprehensive protein interaction analysis:

• Proximity labeling approaches: Combine with BioID or APEX2 approaches by fusing promiscuous biotin ligases to SPAC22A12.08c and using the antibody to verify expression and localization.

• Cross-linking mass spectrometry: Use formaldehyde or DSS cross-linking followed by immunoprecipitation with SPAC22A12.08c antibody to capture transient interactions.

• Yeast two-hybrid validation: Confirm interactions identified through co-IP by independent methods.

• Functional validation: Use genetic interaction studies (synthetic lethality, suppressor screens) to validate biological significance of identified interactions.

What strategies exist for quantifying SPAC22A12.08c expression levels across different experimental conditions?

For accurate protein quantification:

• Western blot quantification methodology:

  • Use internal loading controls (e.g., tubulin, actin)

  • Employ fluorescent secondary antibodies for wider linear range

  • Create standard curves using recombinant protein

  • Utilize image analysis software with background subtraction

• Immunohistochemistry with quantitative image analysis:
  Similar to approaches used for human tissues , develop quantification methods for intensity and distribution patterns.

• Mass spectrometry-based quantification:
  Use antibody for immunoprecipitation followed by targeted MS approaches like PRM or SRM.

How can researchers address epitope masking issues when SPAC22A12.08c antibody shows inconsistent detection patterns?

Epitope masking can occur due to protein-protein interactions, post-translational modifications, or conformational changes:

• Epitope retrieval techniques:

  • Heat-induced epitope retrieval: 10 mM citrate buffer (pH 6.0), 95°C for 20 minutes

  • Enzymatic retrieval: 0.1% trypsin in PBS for 10-15 minutes at 37°C

  • Detergent treatment: 0.5% SDS for 5 minutes at room temperature before blocking

• Denaturing conditions: For Western blots, compare reducing vs. non-reducing conditions and different detergents.

• Cell cycle-dependent modifications: Synchronize cultures to determine if detection varies throughout the cell cycle.

• Protein complex dissociation: Use more stringent lysis conditions or competitive elution strategies to disrupt protein complexes that might mask the epitope.

What methodological approaches can improve signal detection when working with low abundance proteins like SPAC22A12.08c?

For detecting low abundance proteins:

• Signal amplification systems:

  Method	Amplification Factor	Best For
  Tyramide Signal Amplification	10-100×	Immunofluorescence
  Poly-HRP conjugated secondaries	5-20×	Western blot, ELISA
  Biotin-streptavidin systems	3-10×	Multiple applications
  Quantum dots	5-20×	Fluorescence imaging

• Cell fractionation: Enrich for specific cellular compartments where SPAC22A12.08c may be concentrated.

• Optimized immunoprecipitation: Use larger volumes of starting material and optimized antibody concentrations.

• Highly sensitive detection methods: Consider using more sensitive detection reagents similar to those used for human antibody characterization .