SPAC9G1.10c Antibody

What is SPAC9G1.10c and why is it significant in fission yeast research?

SPAC9G1.10c is a gene coding for Sup11p, a protein in Schizosaccharomyces pombe (fission yeast) that plays an essential role in cell wall formation and β-1,6-glucan synthesis. This protein is particularly significant because it is involved in critical cellular processes including septum formation and cell wall integrity. Sup11p shows significant homology to Saccharomyces cerevisiae Kre9, which participates in β-1,6-glucan synthesis, though the precise function mechanism remains under investigation. As an essential gene, sup11+ deletion is lethal, highlighting its biological importance in yeast cell viability .

What are the key structural and functional properties of the Sup11p protein?

Sup11p is a membrane protein that resides in the late Golgi or post-Golgi compartments. It features a signal anchor sequence that orients the protein with its functional domain facing the lumen. In silico analysis indicates that Sup11p is a key component in β-1,6-glucan synthesis pathways. Structurally, it undergoes O-mannosylation post-translational modification, which affects its function. Depletion studies demonstrate that Sup11p is essential for correct septum formation, as mutants with reduced sup11+ expression show severe morphological defects including misshapen septa with abnormal cell wall material accumulation .

What are the optimal experimental design strategies for SPAC9G1.10c antibody studies?

When designing experiments using SPAC9G1.10c antibodies, researchers should implement a balanced and well-controlled approach. First, include appropriate negative controls from wild-type strains alongside positive controls from strains with altered Sup11p expression. Incorporate biological replicates (minimum n=3) to account for biological variability and technical replicates to assess methodological precision. For immunoblotting experiments, use gradient gels (10-15%) to optimize separation of Sup11p, which may undergo post-translational modifications. When performing immunolocalization, compare both chemical fixation and cryofixation methods, as membrane proteins can show fixation artifacts. For co-immunoprecipitation studies investigating Sup11p interactions with cell wall synthesis machinery, use gentle detergents like digitonin to preserve protein complexes. Finally, implement proper normalization using housekeeping proteins from the same cellular compartment as Sup11p .

How should researchers approach antibody validation for SPAC9G1.10c studies?

A robust validation strategy for SPAC9G1.10c antibodies requires multiple complementary approaches. Begin with Western blot analysis using both wild-type samples and those from Sup11p-depleted strains (such as the nmt81-sup11 conditional mutant) to confirm specificity. Conduct immunoprecipitation followed by mass spectrometry to verify that the antibody captures the correct target. Perform subcellular fractionation via sucrose density gradient centrifugation to confirm that the antibody recognizes Sup11p in the expected Golgi/post-Golgi fractions. For immunofluorescence applications, validate antibody specificity using tagged versions of Sup11p (with HA or GFP tags) and compare antibody signals with fluorescent protein localization. Additionally, perform enzyme-linked immunosorbent assays (ELISAs) with purified recombinant Sup11p protein to establish binding affinities and detection limits. Cross-reactivity testing against related proteins should also be conducted to ensure specificity .

What normalization procedures are recommended for antibody microarray studies involving Sup11p?

For antibody microarray studies involving Sup11p detection, implement a comprehensive normalization strategy to minimize technical variation while preserving biological differences. Begin with spatial normalization to correct for position-dependent effects on the array. Apply a global intensity normalization using housekeeping proteins expressed across all experimental conditions. For two-color arrays, use a dye-swap design to control for dye-specific biases, where each sample is labeled with both fluorophores in separate arrays, then averaged. Include eight independent negative controls and at least one positive control sample for normalization of area under the curve values in all experiments, similar to methods used in antibody response studies. For internal normalization, calculate the ratio of Sup11p signal to total protein signal using a reliable method like SYPRO Ruby staining. Additionally, implement quantile normalization to ensure comparable distributions across arrays, followed by log transformation of intensity values to approximate normal distribution, which facilitates downstream statistical analysis .

How can researchers distinguish between specific and non-specific binding when using SPAC9G1.10c antibodies?

To distinguish between specific and non-specific binding when using SPAC9G1.10c antibodies, researchers should implement a multi-faceted approach. First, perform competitive binding assays using purified recombinant Sup11p protein at increasing concentrations to demonstrate signal reduction proportional to competitor concentration. Compare signals between wild-type samples and samples from Sup11p-depleted mutants (such as nmt81-sup11) to identify specific binding patterns. Utilize a Proteinase K protection assay to verify the topology of the protein and confirm antibody epitope accessibility. For immunofluorescence studies, pre-absorb antibodies with recombinant antigen and use this as a control to identify non-specific signals. Implement Western blot analysis with both reducing and non-reducing conditions to verify that the antibody recognizes the correct epitope configuration. Additionally, signal quantification should include background subtraction using areas adjacent to cells or from Sup11p-depleted samples. Cross-validation using multiple antibodies targeting different epitopes of Sup11p can provide confirmation of specific binding patterns .

How can researchers interpret contradictory results between protein detection methods when studying Sup11p?

When faced with contradictory results between protein detection methods for Sup11p, implement a systematic troubleshooting and reconciliation approach. First, evaluate each method's strengths and limitations: Western blots excel at molecular weight determination but may miss conformational epitopes, while immunoprecipitation preserves protein interactions but can introduce artifacts. Consider that Sup11p's membrane localization and post-translational modifications (particularly O-mannosylation) may affect antibody accessibility differently across methods. Examine potential technical variables including sample preparation differences (denaturation conditions, detergent types, buffer compositions) that might alter Sup11p epitope exposure. Investigate whether discrepancies correlate with specific cellular conditions that could affect Sup11p's conformation or interaction partners. Perform fractionation studies to determine if contradictions arise from differential detection of Sup11p populations in distinct subcellular compartments. When methods disagree on Sup11p quantity, implement orthogonal approaches such as mass spectrometry for absolute quantification. Finally, consider that Sup11p interacts with the cell wall and may partition differently between soluble and insoluble fractions, potentially explaining method-specific detection variations .

How can SPAC9G1.10c antibodies be optimized for studying protein-glucan interactions in the fission yeast cell wall?

To optimize SPAC9G1.10c antibodies for studying protein-glucan interactions, develop specialized protocols that preserve the native cell wall architecture. Begin by implementing cell wall biotinylation techniques similar to those described in previous research to tag accessible proteins while maintaining their structural context. Modify fixation protocols to include electron microscopy-grade glutaraldehyde at low concentrations (0.1-0.2%) to cross-link proteins to adjacent glucans while preserving antibody epitopes. For immunoelectron microscopy, use ultra-thin cryosections and immunogold labeling with anti-Sup11p antibodies followed by secondary detection of β-1,6-glucan using specific carbohydrate-binding modules. Develop in situ proximity ligation assays by combining Sup11p antibodies with labeled lectins specific for β-1,6-glucan to visualize close associations. For biochemical studies, implement sequential enzymatic digestions of cell walls with specific glucanases while monitoring Sup11p liberation using quantitative immunoblotting. Additionally, develop pull-down assays using recombinant Sup11p domains to capture interacting glucan fragments, followed by structural characterization using mass spectrometry and NMR .

What techniques can researchers employ to study the dynamics of Sup11p localization during cell cycle progression?

To study Sup11p localization dynamics throughout the cell cycle, implement complementary live and fixed-cell approaches. Develop stable S. pombe strains expressing fluorescently-tagged Sup11p (overcoming previous tagging challenges) using split-fluorescent protein systems or small epitope tags combined with high-affinity nanobodies. Synchronize cells using cdc25-22 temperature-sensitive mutants or lactose gradient centrifugation, then collect samples at defined cell cycle stages. Implement four-dimensional imaging (x, y, z, time) with spinning disk confocal microscopy to track Sup11p movement with minimal phototoxicity. For quantitative analysis, develop computational pipelines that segment cells based on septum formation and measure Sup11p distribution relative to cell cycle markers. Combine this with photoactivatable fluorescent protein fusions to measure Sup11p mobility and trafficking rates between compartments. For higher resolution, apply structured illumination microscopy to visualize Sup11p in relation to Golgi/post-Golgi markers throughout the cell cycle. Additionally, implement correlative light and electron microscopy to connect fluorescence data with ultrastructural context, particularly important during septum formation when Sup11p plays a critical role in β-1,6-glucan deposition .

How can researchers utilize antibody-based approaches to investigate the relationship between Sup11p O-mannosylation and its function?

To investigate the relationship between Sup11p O-mannosylation and function, develop specialized antibody-based approaches combined with glycobiology techniques. Generate and characterize antibodies that specifically recognize either O-mannosylated or non-mannosylated forms of Sup11p using synthetic peptides representing known modification sites. Establish quantitative immunoblotting protocols that can distinguish these forms using O-mannosylation-specific antibodies alongside total Sup11p detection. Implement immunoprecipitation of Sup11p followed by lectin blotting using ConA (mannose-specific) and compare signals across wild-type cells versus O-mannosyl transferase mutants (oma2, oma4). For functional studies, develop pull-down assays using differentially mannosylated recombinant Sup11p domains to identify interaction partners that prefer specific glycoforms. Apply proximity-dependent biotinylation (BioID) with Sup11p fusions in both wild-type and O-mannosylation-deficient backgrounds to map changes in the protein's interactome. For structural studies, use antibodies to immunopurify native Sup11p from different genetic backgrounds for glycopeptide mapping by mass spectrometry to create a comprehensive O-mannosylation site map. Finally, develop assays that correlate Sup11p O-mannosylation levels with β-1,6-glucan synthesis rates using antibodies to monitor both processes simultaneously .

What strategies can address poor signal-to-noise ratios when using SPAC9G1.10c antibodies in fission yeast?

To address poor signal-to-noise ratios when using SPAC9G1.10c antibodies, implement a systematic optimization approach. Begin by modifying sample preparation: for cell wall-associated proteins like Sup11p, test different spheroplasting methods using various concentrations of zymolyase or lysing enzymes to improve antibody accessibility while maintaining protein integrity. Optimize blocking conditions by comparing BSA, casein, and commercial blocking reagents at different concentrations (3-5%) and extended blocking times (2-4 hours) to reduce non-specific binding. For immunofluorescence, implement sample clearing techniques using glycerol-based mounting media with anti-fade agents to reduce autofluorescence from cell wall components. Test signal amplification methods such as tyramide signal amplification or rolling circle amplification to enhance specific signals while maintaining low background. For Western blots, optimize transfer conditions specifically for membrane proteins using mixed transfer buffers (SDS and alcohols) and longer transfer times at lower voltages. Additionally, implement batch pre-adsorption of antibodies against wild-type cell lysates from Sup11p-depleted strains to remove cross-reactive antibodies. Finally, compare different detection systems (chemiluminescence, fluorescence, colorimetric) to identify the optimal signal-to-noise ratio for each application .

How can researchers overcome cross-reactivity issues with other cell wall proteins when using Sup11p antibodies?

To overcome cross-reactivity issues with other cell wall proteins when using Sup11p antibodies, implement a multi-faceted specificity enhancement strategy. Begin by performing epitope mapping to identify unique Sup11p sequences with minimal homology to other cell wall proteins, then generate new antibodies against these regions or affinity-purify existing polyclonal antibodies using the unique peptide sequences. Implement competitive Western blotting using recombinant fragments of potential cross-reactive proteins to identify and characterize specific cross-reactivities. For immunoprecipitation experiments, develop two-step immunopurification protocols using antibodies targeting different Sup11p epitopes to dramatically reduce non-specific capture. When working with cell wall preparations, implement sequential extraction procedures that separate proteins based on their linkage to different cell wall components (β-1,3-glucan vs. β-1,6-glucan), enriching for Sup11p-specific fractions before antibody application. For immunofluorescence, use multi-color imaging with known markers of potential cross-reactive proteins to identify and exclude false positive signals. Additionally, validate all findings using genetic approaches such as epitope-tagged Sup11p expressed in sup11Δ backgrounds complemented with an inducible sup11+ copy to create unambiguous positive and negative controls .

What approaches help ensure reproducibility in quantitative Sup11p expression studies across different laboratories?

To ensure reproducibility in quantitative Sup11p expression studies across different laboratories, implement standardized protocols and reference materials. Develop and distribute a common set of calibrated recombinant Sup11p protein standards at defined concentrations to serve as absolute quantification references. Establish a shared panel of reference S. pombe strains with characterized Sup11p expression levels, including wild-type, tagged, and conditional expression mutants like nmt81-sup11. Create detailed standard operating procedures covering all aspects from cell growth conditions (specific media compositions, OD600 harvesting points) to lysis methods optimized for membrane protein extraction and antibody incubation parameters. Implement data normalization using multiple housekeeping proteins validated for stability across experimental conditions relevant to Sup11p studies. For antibody-based quantification, establish inter-laboratory validation studies using the same antibody lots or develop monoclonal antibodies with defined epitope specificity to eliminate polyclonal batch variations. Additionally, create open-access databases for sharing raw data and analysis workflows that include standardized methods for image processing, signal quantification, and statistical analysis. Finally, implement regular cross-laboratory proficiency testing using identical samples analyzed with local protocols to identify sources of variability .

How might emerging antibody technologies advance our understanding of Sup11p function in cell wall biosynthesis?

Emerging antibody technologies offer transformative potential for understanding Sup11p's role in cell wall biosynthesis. Single-domain nanobodies derived from camelid antibodies could provide superior access to sterically restricted epitopes within the cell wall matrix, enabling in vivo tracking of Sup11p with minimal interference to protein function. Proximity-dependent labeling using antibody-enzyme fusions (such as APEX2 or TurboID) could map the dynamic Sup11p interactome during different phases of cell wall synthesis, revealing transient protein and polysaccharide interactions. DNA-barcoded antibodies used in spatial transcriptomics workflows could simultaneously quantify and localize Sup11p together with hundreds of other proteins involved in cell wall biosynthesis, creating comprehensive spatial interaction maps. Super-resolution techniques like DNA-PAINT, using antibody-oligonucleotide conjugates, could achieve nanometer-scale resolution of Sup11p organization within the cell wall synthesis machinery. Antibody-directed CRISPR systems could enable precise manipulation of Sup11p in specific subcellular locations to test spatial-dependent functions. Finally, synthetic antibody-based biosensors could be developed to detect conformational changes in Sup11p associated with its catalytic activity in β-1,6-glucan synthesis, providing real-time activity measurements rather than simple localization data .

What research questions about SPAC9G1.10c remain unresolved and how might antibody-based approaches address them?

Several critical questions about SPAC9G1.10c/Sup11p remain unresolved and could be addressed through innovative antibody-based approaches. First, the precise molecular mechanism by which Sup11p contributes to β-1,6-glucan synthesis remains unknown—developing conformation-specific antibodies that recognize Sup11p in different catalytic states could help elucidate this mechanism. Second, the complete interactome of Sup11p during cell wall synthesis is undefined; implementing proximity-dependent biotinylation with Sup11p-antibody conjugates could identify interaction partners in their native context. Third, the temporal and spatial coordination between Sup11p activity and other cell wall synthesis enzymes during septum formation remains poorly understood; multi-color super-resolution microscopy with phase-specific antibodies could map this coordination process. Fourth, the regulatory mechanisms controlling Sup11p activity and localization during the cell cycle need clarification; developing phospho-specific antibodies could identify key regulatory modifications. Fifth, the evolutionary conservation of Sup11p function across fungal species remains incompletely characterized; creating cross-species reactive antibodies against conserved domains could enable comparative studies. Finally, the potential of Sup11p as an antifungal target requires exploration; antibodies recognizing accessible epitopes in live cells could help evaluate its druggability and guide development of therapeutic strategies against pathogenic fungi with Sup11p homologs .

Comparative Analysis of Sup11p Detection Methods

Key Phenotypic Changes in Sup11p-Depleted Cells

The depletion of Sup11p in S. pombe results in significant cell wall remodeling processes, as evidenced by transcriptomic analysis. Cells with reduced Sup11p expression show severe morphological defects, particularly in septum formation. The central region of the septum contains abnormal accumulations of cell wall material, including β-1,3-glucan, which is typically confined to the primary septum. Analysis of the nmt81-sup11 mutant reveals that Gas2p (a glucanase) plays a significant role in the development of the septum phenotype. Most critically, Sup11p is essential for β-1,6-glucan synthesis, as mutants with reduced expression show no detectable β-1,6-glucan in the cell wall .