SPCC162.02c Antibody

What cellular functions does the SPCC162.02c gene product (Sup11p) perform in fission yeast?

Sup11p is an essential protein localized to the Golgi apparatus and plasma membrane that plays critical roles in:

• Cell wall integrity maintenance

• Cytokinesis and proper septum formation

• β-1,6-glucan synthesis pathways

• Septum assembly

The protein is indispensable for proper septum assembly, with mutants showing severe morphological defects and malformation of the septum with massive accumulation of cell wall material . Specifically, Sup11p functions as a key component in β-1,6-glucan synthesis, as evidenced by the absence of β-1,6-glucan from the cell wall in a conditionally lethal nmt81-sup11 knock-down mutant .

Sup11p shows significant homology to Saccharomyces cerevisiae Kre9, which is involved in β-1,6-glucan synthesis . Importantly, genetic interaction studies have demonstrated that Sup11p interacts with glucanase family members, suggesting a coordinated role in cell wall remodeling .

How is SPCC162.02c expression regulated, and what are the implications for antibody-based detection methods?

Transcriptome analysis performed on the nmt81-sup11 mutant identified significant regulation of several cell wall glucan modifying enzymes, indicating a complex regulatory network . When designing antibody-based detection methods, researchers should consider:

• Transcriptional variation under different growth conditions

• Post-translational modifications affecting epitope accessibility

• Protein localization changes during cell cycle

In the nmt81-sup11 depletion mutant, there are observable variations in cell wall protein composition, which suggests that Sup11p levels influence the expression or localization of other cell wall proteins . This finding has implications for experimental design when using antibodies to study Sup11p in different genetic backgrounds.

What methodological approaches are most effective for developing antibodies against yeast cell wall proteins like Sup11p?

Developing antibodies against yeast cell wall proteins presents unique challenges due to their glycosylation and membrane association. Based on successful approaches with similar proteins, researchers should consider:

• Antigen selection strategies:

  • Recombinant protein expression in E. coli (as demonstrated with SHC1 immunogen preparation)

  • Synthetic peptides corresponding to specific domains

  • Transmembrane topology analysis to identify accessible epitopes

• Expression systems for antigen production:

  • For Sup11p, an E. coli-derived recombinant protein approach similar to that used for SHC1 antibody development may be effective

• Purification methods:

  • Affinity chromatography on Protein G for IgG purification

  • Buffer conditions suitable for membrane proteins (phosphate buffered saline with appropriate preservatives)

Cell wall biotinylation and antigen purification methods have been successfully employed with yeast proteins, as referenced in the Sup11p characterization study . When developing antibodies against Sup11p, researchers may also consider the polyclonal antibody approach using GST-fusion peptides, which was successful in the original Sup11p study .

What validation experiments are essential to confirm SPCC162.02c antibody specificity and performance?

Thorough validation is critical for antibodies targeting yeast proteins. Based on established protocols for similar proteins, researchers should perform:

• Western blot analysis:

  • Should detect bands at the expected molecular weight (~30.6 kDa for Sup11p)

  • Compare wild-type and sup11+ mutant strains to confirm specificity

  • Include positive controls with tagged versions of Sup11p (e.g., HA-tagged constructs as used in original studies)

• Immunoprecipitation validation:

  • Verify ability to capture the target protein from cell lysates

  • Confirm identity of precipitated proteins by mass spectrometry

• Cross-reactivity testing:

  • Test against related species or homologous proteins

  • Evaluate performance across different sample preparation methods

• Reproducibility assessment:

  • Document coefficient of variation (CV) across multiple experiments

  • Based on standards for similar antibodies, aim for CV values of <15% (comparable to the 9.8-14.4% range reported for anti-p16 IgG assays)

When validating an antibody against SPCC162.02c, researchers should include controls similar to those used in the PrecisionAb antibody validation program referenced in search result , which features defined performance criteria and batch-to-batch consistency testing.

What are the optimal experimental conditions for using SPCC162.02c antibodies in immunolocalization studies?

Based on subcellular localization studies of Sup11p and similar proteins, researchers should consider:

• Sample preparation protocols:

  • For immunofluorescence: Spheroplasting of S. pombe using enzymes that digest the cell wall is critical

  • For immunoelectron microscopy: Appropriate fixation to preserve antigen accessibility while maintaining cellular ultrastructure

• Optimization parameters:

  • Antibody dilution: Starting with 1/1000 dilution (similar to SHC1 antibody protocols)

  • Incubation conditions: Time and temperature optimization

  • Blocking agents: BSA or serum appropriate for secondary antibody species

• Controls to include:

  • Positive controls: HA-tagged Sup11p constructs

  • Negative controls: sup11 deletion or depletion strains

  • Secondary antibody-only controls to assess background

The search results indicate that Sup11p localizes to late Golgi or post-Golgi compartments and plasma membrane . Researchers should use antibodies against known markers of these compartments (e.g., Golgi markers) for co-localization studies to confirm specificity of staining patterns.

How can SPCC162.02c antibodies be effectively used in Western blotting protocols?

For optimal Western blotting with SPCC162.02c antibodies, researchers should adapt protocols that have worked for similar yeast proteins:

• Sample preparation considerations:

  • Cell lysis buffers compatible with membrane proteins

  • Proteinase K protection assay for topology analysis

  • EndoH treatment to assess glycosylation status

• Electrophoresis and transfer parameters:

  • SDS-PAGE conditions optimized for membrane proteins

  • Transfer conditions appropriate for the molecular weight of Sup11p (~30.6 kDa)

• Detection optimizations:

  • Dilution range: Start with 1/1000 as established for similar antibodies

  • Signal enhancement: Consider using ECL systems suitable for low abundance proteins

  • Stripping and reprobing: Protocols for membrane regeneration if multiple proteins need to be detected

Based on the Sup11p localization studies, researchers should be aware that post-translational modifications, particularly O-mannosylation, may affect the apparent molecular weight in SDS-PAGE . Consider treating samples with glycosidases to confirm identity of bands if multiple signals are observed.

How can SPCC162.02c antibodies be used to investigate the role of Sup11p in β-1,6-glucan synthesis?

To investigate Sup11p's role in β-1,6-glucan synthesis, researchers can employ the following advanced applications:

• Protein complex analysis:

  • Co-immunoprecipitation with SPCC162.02c antibodies to identify Sup11p interaction partners

  • Analysis of precipitated complexes by mass spectrometry

  • Validation of interactions with known components of glucan synthesis pathways

• Functional pathway analysis:

  • Combine antibody-based detection with genetic approaches targeting interacting genes

  • Compare β-1,6-glucan content in mutant strains using specific labeling methods

  • Analyze the data in context of the genetic interactions with β-1,6-glucanase family members identified in previous studies

• Temporal regulation studies:

  • Cell cycle synchronization combined with antibody-based detection to monitor Sup11p levels and localization

  • Correlation with septum formation phases

Research findings reveal that Sup11p is required for β-1,6-glucan formation and proper septum assembly . Furthermore, Gas2p (a member of the β-1,3-glucanosyl-transferases GH72 family) plays a crucial role in accumulating septum material depositions in the nmt81-sup11 mutant . This relationship should be considered when designing experiments using SPCC162.02c antibodies to study β-1,6-glucan synthesis.

What methodological considerations are important when using SPCC162.02c antibodies to study protein-protein interactions?

When investigating protein-protein interactions involving Sup11p, researchers should consider:

• Immunoprecipitation strategies:

  • Cell lysis conditions that preserve native protein interactions

  • Cross-linking approaches to capture transient interactions

  • Detergent selection critical for membrane protein solubilization

• Alternative methods to validate interactions:

  • Proximity ligation assays to detect protein interactions in situ

  • FRET/BRET approaches with fluorescently tagged proteins

  • Yeast two-hybrid or split-ubiquitin systems for membrane proteins

• Experimental controls:

  • IgG control immunoprecipitations

  • Competitive binding with excess antigen

  • Reciprocal co-immunoprecipitation with antibodies against putative interaction partners

Recent studies have demonstrated that Sup11p genetically interacts with glucanase family genes (e.g., gas2+), providing potential targets for protein-protein interaction studies . The table below summarizes key Sup11p findings that may guide interaction studies:

What are common challenges when using antibodies against yeast membrane proteins like Sup11p, and how can they be addressed?

Researchers working with yeast membrane proteins face several technical challenges:

• Accessibility issues:

  • Challenge: Epitope masking due to protein topology or post-translational modifications

  • Solution: Use multiple antibodies targeting different regions; consider enzymatic treatments to expose epitopes

• Non-specific binding:

  • Challenge: High background in yeast samples due to cross-reactivity

  • Solution: Optimize blocking conditions; pre-absorb antibodies with wild-type lysates from control strains

• Signal variability:

  • Challenge: Inconsistent staining patterns between experiments

  • Solution: Standardize fixation and permeabilization protocols; include internal controls in each experiment

• Post-translational modifications:

  • Challenge: Variable glycosylation affecting antibody recognition

  • Solution: Compare results with and without deglycosylation treatments; use antibodies against protein backbone rather than modified regions

The research on Sup11p demonstrated that it undergoes O-mannosylation, and when expressed in an O-mannosylation mutant background, it can be hypo-mannosylated and even undergo unusual N-glycosylation on an N-X-A sequon . This highlights the importance of considering post-translational modifications when interpreting antibody-based detection results.

How can researchers interpret contradictory results when using SPCC162.02c antibodies in different experimental contexts?

When faced with contradictory results:

• Systematic validation approach:

  • Verify antibody specificity in each experimental system

  • Compare results from multiple detection methods (Western blot, immunofluorescence, ELISA)

  • Use genetic controls (tagged versions, deletion mutants) to confirm specificity

• Technical considerations:

  • Evaluate sample preparation variations (lysis methods, fixation protocols)

  • Assess antibody lot-to-lot variability

  • Consider epitope accessibility in different experimental contexts

• Biological interpretations:

  • Protein expression levels may vary with growth conditions

  • Post-translational modifications may differ between conditions

  • Protein localization changes during cell cycle

When interpreting results from SPCC162.02c antibody experiments, researchers should be aware of the protein's dual localization to the Golgi apparatus and plasma membrane. This dynamic localization may result in apparently contradictory results depending on the experimental conditions and cell cycle stage of the samples being analyzed.