SPAC22A12.06c Antibody

What is SPAC22A12.06c protein and what are its key cellular functions?

SPAC22A12.06c (Sup11p) is an essential protein in Schizosaccharomyces pombe (fission yeast) involved in several critical cellular processes:

The protein localizes primarily to the Golgi/post-Golgi apparatus, consistent with its role in cell wall component synthesis and trafficking.

What experimental applications is the SPAC22A12.06c antibody validated for?

The SPAC22A12.06c antibody has been validated for the following applications:

• Western Blot (WB): Confirmed to detect antigen-specific bands in fission yeast lysates .

• ELISA: Demonstrates high titer sensitivity, with effective detection at dilutions up to 1:64,000 .

• Specificity Testing: Recognizes recombinant SPAC22A12.06c protein without cross-reactivity to unrelated antigens.

What are the recommended storage and handling conditions for the SPAC22A12.06c antibody?

For optimal performance and longevity of the SPAC22A12.06c antibody:

• Storage Temperature: Store at -20°C or -80°C upon receipt .

• Avoid Freeze-Thaw Cycles: Repeated freezing and thawing should be minimized as it can compromise antibody activity .

• Storage Buffer: The antibody is supplied in a stabilizing buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative .

• Stability: Maintains activity in the provided storage buffer for >12 months when stored at recommended temperatures.

How is the SPAC22A12.06c antibody produced and purified?

The SPAC22A12.06c antibody production and purification process involves:

• Immunogen: Generated using recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) SPAC22A12.06c protein .

• Host Species: Raised in rabbits .

• Clonality: Polyclonal antibody, recognizing multiple epitopes of the target protein .

• Isotype: IgG .

• Purification Method: Antigen affinity purification to ensure high specificity and minimal background reactivity .

• Form: Supplied as a liquid preparation .

What is the species reactivity profile of the SPAC22A12.06c antibody?

The SPAC22A12.06c antibody has been specifically validated for:

• Primary Reactivity: Schizosaccharomyces pombe (strain 972/ATCC 24843) .

• Cross-Reactivity: While primarily designed for S. pombe, potential cross-reactivity with homologous proteins in closely related fungi may exist, though this would require experimental validation.

• Homolog Recognition: The antibody may detect the S. cerevisiae homolog Kre9 due to conserved domains, though specificity testing would be necessary to confirm this potential application.

How can the SPAC22A12.06c antibody be utilized to study β-1,6-glucan synthesis in fission yeast?

The SPAC22A12.06c antibody provides several powerful approaches to investigate β-1,6-glucan synthesis:

• Protein Localization Studies: Immunofluorescence or immunoelectron microscopy using the antibody can track the spatial distribution of Sup11p in relation to sites of active β-1,6-glucan synthesis.

• Co-Immunoprecipitation Assays: The antibody can be used to identify protein complexes associated with Sup11p, potentially revealing other components of the β-1,6-glucan synthesis machinery.

• Correlation Analysis: Western blotting with this antibody, combined with quantitative β-1,6-glucan measurements, can establish relationships between Sup11p expression levels and glucan production.

• Pulse-Chase Experiments: The antibody can be used to monitor Sup11p turnover rates in relation to cell wall synthesis dynamics.

Research has demonstrated that SPAC22A12.06c (Sup11p) is indispensable for β-1,6-glucan polymer formation, with knockdown mutants exhibiting complete absence of this polysaccharide and consequent cell wall fragility.

What is the relationship between SPAC22A12.06c (Sup11p) and septum formation during cell division?

SPAC22A12.06c plays crucial roles in septum development and integrity:

• Primary and Secondary Septum Formation: Studies using Ags1 (another glucan synthase that co-localizes with Sup11p) demonstrate that proper glucan synthesis is essential for both primary septum structural strength and secondary septum formation .

• Septum Strength: The antibody can be used to investigate how Sup11p contributes to septum structural integrity, particularly important during the physical stress of cell separation .

• Collaboration with CAR Assembly: Research indicates that proper localization of cell wall synthesis proteins like Sup11p depends on the contractile actomyosin ring (CAR) formation and positioning .

• Experimental Approach: Time-lapse microscopy combined with immunolabeling can track the dynamic relationship between Sup11p localization and septum formation.

Depletion of such cell wall synthesis proteins causes malformed septa with aberrant accumulation of β-1,3-glucan, critically disrupting cytokinesis .

How does SPAC22A12.06c interact with Bgs1 in septum construction?

The interaction between SPAC22A12.06c and Bgs1 (another glucan synthase) represents a critical collaboration in cell wall synthesis:

• Co-localization Evidence: Studies show tight co-localization of Ags1 (a glucan synthase similar to Sup11p) with Bgs1, suggesting cooperation in early steps of septum construction .

• Genetic Interaction: There is genetic evidence for interaction between glucan synthases, as the double mutant cps1-12 mok1-664 shows greater temperature sensitivity than single mutants .

• Compensatory Mechanisms: Interestingly, defective Bgs1 function can suppress the lytic phenotype of Ags1 absence, suggesting complex interactions in cell wall integrity pathways .

• Synthetic Phenotypes: The combined absence of both synthases produces severe cytokinesis defects not observed with single protein absence .

What post-translational modifications affect SPAC22A12.06c function?

SPAC22A12.06c undergoes several functionally significant post-translational modifications:

• O-Mannosylation: Research indicates that hypo-mannosylated Sup11p in oma4Δ mutants undergoes atypical N-glycosylation at a sequon normally masked by O-mannose residues.

• Glycosylation Patterns: The antibody can be used to immunoprecipitate Sup11p for glycoproteomic analysis to map exact modification sites.

• Functional Consequences: Altered glycosylation patterns are associated with changes in protein localization, stability, and enzymatic activity.

• Methodological Approach: Researchers can use enzymatic deglycosylation followed by Western blotting with the SPAC22A12.06c antibody to determine the extent of glycosylation and its impact on protein function.

How can the SPAC22A12.06c antibody contribute to antifungal drug development research?

The SPAC22A12.06c antibody offers valuable tools for antifungal drug research:

• Target Validation: The antibody can confirm whether candidate drugs affect Sup11p levels, localization, or post-translational modifications.

• Mechanism of Action Studies: Combined with viability assays, the antibody can help determine if cell death correlates with disruption of Sup11p function.

• Resistance Mechanisms: In drug-resistant strains, the antibody can assess whether Sup11p expression or modification patterns have changed.

• Screening Platform: Development of high-throughput ELISA using this antibody could screen compound libraries for those affecting Sup11p.

This is particularly relevant as fungal cell wall components like β-1,6-glucan are absent in mammalian cells, making them excellent targets for selective antifungal therapies .

What experimental approaches can be used to study SPAC22A12.06c localization in fission yeast cells?

Several sophisticated techniques can be employed to study SPAC22A12.06c localization:

• Immunofluorescence Microscopy: Using the SPAC22A12.06c antibody for fixed cell imaging.

• Live-Cell Imaging: Using a physiological Ags1-GFP variant (similar to SPAC22A12.06c) to track protein dynamics in real-time .

• Correlative Microscopy: Combining fluorescence microscopy with electron microscopy for nanoscale resolution.

• Co-localization Studies: Dual-labeling with the SPAC22A12.06c antibody and markers for cellular compartments like the Golgi apparatus.

Research has revealed that Ags1 (similar to Sup11p) co-localizes with other glucan synthases like Bgs1 at growing cell tips, division sites, and in all sites of wall synthesis during sexual differentiation, including mating, spore formation, and spore germination .

How does SPAC22A12.06c function compare with its homologs in other fungi?

Comparative analysis reveals important functional relationships between SPAC22A12.06c and its fungal homologs:

Key differences and similarities include:

• Functional Conservation: Both proteins are involved in β-1,6-glucan synthesis, though Sup11p appears to have a more critical role in S. pombe than Kre9 does in S. cerevisiae.

• Localization Differences: While both proteins are found in the secretory pathway, their precise subcellular distribution differs between species.

• Experimental Approaches: The SPAC22A12.06c antibody could potentially be used in cross-species studies to detect conserved epitopes in homologous proteins, though this would require validation.

What tools are available for genome manipulation studies involving SPAC22A12.06c?

Several sophisticated genetic tools have been developed for studying SPAC22A12.06c:

• I-PpoI Endonuclease System: A tetracycline-inducible system producing rapid cleavage (>80% after 1 hour) at defined genomic sites, allowing controlled introduction of double-strand breaks for studying DNA repair processes .

• Genome Reduction Studies: Large-scale gene deletion approaches have been used in S. pombe to identify minimal gene sets required for growth, with some strains having deletions spanning 657.3 kb (223 genes) .

• Site-Specific Double Strand Break Assays: These systems overcome limitations of ionizing radiation approaches by introducing breaks at defined genomic locations .

• Physiological Protein Tagging: Creation of functional tagged variants like Ags1-GFP that maintain protein activity while allowing visualization .

These tools provide researchers with precise methods to manipulate SPAC22A12.06c expression and function in experimental settings.

How does SPAC22A12.06c depletion affect cell wall integrity and transcriptional responses?

SPAC22A12.06c depletion triggers multiple cellular responses:

• Transcriptional Regulation: Sup11p depletion upregulates glucan-modifying enzymes (e.g., Gas2p), indicating compensatory cell wall remodeling processes.

• Cell Lysis Phenotype: After 3 hours of ags1+ repression, lysis occurs at the septum-cell wall border in 13% of cells, typically affecting only one daughter cell. By 7 hours, this progresses to 41% of cells with lysis on both sides of the septum-cell wall border .

• Growth Conditions: Adding osmotic stabilizers like sorbitol can delay cell lysis, providing an experimental window to study early effects of protein depletion .

• Polarized Growth Effects: In some genetic backgrounds (like SIN mutants), SPAC22A12.06c depletion can cause lysis at cell pole tips, indicating its role in maintaining cell wall integrity during polarized growth .

Researchers can use the SPAC22A12.06c antibody in time-course experiments following gene repression to monitor protein depletion rates in relation to the emergence of phenotypic defects.

What is the role of SPAC22A12.06c in the SIN pathway and cytokinesis regulation?

SPAC22A12.06c has complex relationships with the Septation Initiation Network (SIN) and cytokinesis:

• CAR Dependency: SPAC22A12.06c localization to the division site depends on proper Contractile Actomyosin Ring (CAR) formation and positioning, but not directly on the SIN pathway .

• SIN Pathway Interactions: Genetic studies show that mok1-664 SIN double mutants are more temperature-sensitive than single mutants, indicating functional interactions .

• Septation Defects: In SIN mutant backgrounds, SPAC22A12.06c mutation increases septum lysis defects during attempted but incomplete septation .

• Cell Cycle Regulation: The SPAC22A12.06c antibody can be used in synchronized cell populations to track protein expression and localization changes through the cell cycle.