SPAC17A2.07c Antibody

What is SPAC17A2.07c and why is it significant in research?

SPAC17A2.07c is a gene locus in S. pombe that encodes proteins involved in cell wall synthesis and septum formation during cell division. The SPAC17A2.07c antibody targets these proteins, enabling researchers to investigate crucial cellular processes including cell wall integrity maintenance and cytokinesis. This antibody serves as an important tool for understanding fundamental aspects of fungal cell biology that may have implications for antifungal drug development and basic cell biology research.

What are the main cellular functions of the SPAC17A2.07c gene product?

The SPAC17A2.07c gene product is implicated in multiple critical cellular processes:

• Cell Wall Synthesis: Regulates β-1,3-glucan and β-1,6-glucan polymer deposition, essential for maintaining cell wall integrity

• Septum Formation: Involved in the assembly and separation of the septum during cytokinesis, ensuring proper cell division

• Protein Glycosylation: Associates with GPI-anchored proteins and Pir proteins, which are covalently linked to the cell wall matrix

• Cell Wall Dynamics: Upregulated during septum formation, correlating with increased β-glucan synthesis

What is the structure and composition of the SPAC17A2.07c antibody?

The SPAC17A2.07c antibody is likely an IgG isotype immunoglobulin composed of four polypeptide chains: two identical heavy chains (H) and two identical light chains (L). These chains form antigen-binding sites (Fab regions) and an effector region (Fc). The IgG structure provides the high specificity and stability required for applications such as Western blotting and immunoprecipitation.

What laboratory techniques can utilize the SPAC17A2.07c antibody?

The SPAC17A2.07c antibody can be employed in multiple molecular biology techniques as outlined in the table below:

What is the optimal protocol for immunoprecipitation with SPAC17A2.07c antibody?

For effective immunoprecipitation using the SPAC17A2.07c antibody, follow this protocol:

• Sample Preparation: Prepare cell lysates under conditions that preserve protein-protein interactions using an appropriate lysis buffer

• Pre-clearing: Incubate lysate with protein A/G beads to reduce non-specific binding

• Antibody Binding: Add SPAC17A2.07c antibody (typically 2-5 μg per 500 μg of protein) and incubate at 4°C overnight with gentle rotation

• Immunocomplex Capture: Add fresh protein A/G beads and incubate for 1-3 hours at 4°C

• Washing: Perform 3-5 washes with buffer of increasing stringency to remove non-specific binding

• Elution: Elute bound proteins with SDS sample buffer or mild acid elution

• Analysis: Analyze by SDS-PAGE followed by Western blotting or mass spectrometry

This approach has successfully identified interactions between SPAC17A2.07c-encoded proteins and other cell wall components such as Gas2p.

How can the SPAC17A2.07c antibody be optimized for Western blotting?

For optimal Western blotting results with the SPAC17A2.07c antibody:

• Protein Extraction: Use TCA extraction for whole cell extracts from S. pombe to ensure complete protein recovery

• SDS-PAGE Separation: Employ an appropriate percentage gel (typically 8-12%) based on the target protein size

• Transfer Conditions: Use semi-dry or wet transfer methods optimized for fungal proteins

• Blocking: Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary Antibody Incubation: Dilute SPAC17A2.07c antibody (typical range 1:1000-1:5000) and incubate overnight at 4°C

• Detection: Use an HRP-conjugated secondary antibody with appropriate chemiluminescent substrates

• Controls: Include wild-type, knockout, and tagged variants as controls

Western blotting with this antibody has been successfully used to confirm hypo-mannosylation of Sup11p in O-mannosylation mutants .

How does SPAC17A2.07c relate to cell wall dynamics during septum formation?

SPAC17A2.07c plays a critical role in septum formation and cell division in S. pombe. Research indicates:

• The protein is upregulated during septum formation, correlating with increased β-glucan synthesis

• In conditionally lethal nmt81-sup11 knock-down mutants, severe morphological defects and malformation of the septum occur, with massive accumulation of cell wall material at the center of the closing septum

• These depositions consist partially of β-1,3-glucan, which should normally be restricted to the primary septum

• Transcriptome analysis of nmt81-sup11 mutants identified significant regulation of several cell wall glucan modifying enzymes

• The protein likely coordinates with Gas2p, a member of the β-1,3-glucanosyl-transferases GH72 family, which plays a crucial role in accumulating the observed septum material depositions

These findings highlight SPAC17A2.07c's essential role in maintaining proper septum assembly during cell division.

What is the relationship between SPAC17A2.07c and protein glycosylation pathways?

SPAC17A2.07c has a complex relationship with protein glycosylation pathways:

• Sup11p:HA (a tagged version of the protein encoded by SPAC17A2.07c homolog) is itself an O-mannoprotein

• When expressed in an O-mannosylation mutant background, Sup11p:HA is hypo-mannosylated

• Interestingly, in the oma4 mutant background, Sup11p:HA can undergo unusual N-glycosylation on an N-X-A sequon

• This N-X-A sequon is normally located within an S/T-rich region that is heavily O-mannosylated in wild-type yeast, effectively masking the sequon from N-glycosylation machinery

• This suggests competition between O-mannosylation and N-glycosylation pathways for modification sites on SPAC17A2.07c-encoded proteins

This interplay between different glycosylation pathways may be crucial for proper protein function in cell wall biogenesis.

How does the homology between SPAC17A2.07c and S. cerevisiae Kre9 inform functional studies?

SPAC17A2.07c shows significant homology to Saccharomyces cerevisiae Kre9, providing important comparative insights:

• Both proteins are involved in β-1,6-glucan synthesis, though the exact mechanism remains uncertain

• While the proteins share sequence similarity, there are notable structural differences in the β-1,6-glucan polymer between species:

  • In S. pombe, 75% of β-1,6-glucan residues are β-1,3 linked

  • In S. cerevisiae, only 7% are β-1,3 linked

  • In C. albicans, 15% are β-1,3 linked

• The fission yeast β-1,6-glucan backbone is highly branched compared to other yeasts

• These structural differences suggest adaptation of the conserved protein family to species-specific cell wall architectures

• Functional complementation studies between the two homologs could provide insights into conserved and divergent mechanisms

This evolutionary relationship provides a framework for understanding the fundamental mechanisms of β-1,6-glucan synthesis across fungal species.

What controls should be included when using the SPAC17A2.07c antibody?

To ensure experimental validity when using the SPAC17A2.07c antibody, incorporate the following controls:

• Positive Control: Use wild-type S. pombe samples where SPAC17A2.07c protein is known to be expressed

• Negative Control: Include samples from a conditional knock-down strain (e.g., nmt81-sup11) under repressive conditions

• Tagged Control: If available, use strains expressing tagged versions (e.g., SPAC17A2.07c:HA or SPAC17A2.07c:GFP) for specificity verification

• Secondary Antibody-Only Control: Omit primary antibody to detect non-specific binding

• Loading Control: Include detection of a housekeeping protein (e.g., actin or tubulin) for normalization in Western blotting

• Pre-immune Serum Control: For polyclonal antibodies, include pre-immune serum to establish baseline reactivity

These controls help distinguish specific antibody binding from background and non-specific interactions.

How can cross-reactivity issues be addressed when working with SPAC17A2.07c antibody?

To minimize cross-reactivity and improve specificity:

• Optimize Antibody Concentration: Use titration experiments to determine the minimum effective concentration

• Increase Blocking Stringency: Extend blocking time or try alternative blocking agents (BSA, casein, or commercial blockers)

• Modify Washing Conditions: Increase wash duration, frequency, or detergent concentration

• Pre-absorb Antibody: Incubate with lysates from cells lacking the target protein before use in experiments

• Adjust Buffer Composition: Modify salt and detergent concentrations to increase specificity

• Perform Sequential IP: For complex samples, conduct multiple rounds of immunoprecipitation

• Compare Results with Alternative Antibodies: If available, use antibodies targeting different epitopes of the same protein

These approaches can significantly improve signal-to-noise ratio and enhance experimental outcomes.

What are the challenges in studying β-1,6-glucan synthesis using SPAC17A2.07c antibody?

Studying β-1,6-glucan synthesis using SPAC17A2.07c antibody presents several technical challenges:

• Complex Polymer Structure: The highly branched structure of β-1,6-glucan in S. pombe (75% of residues are β-1,3 linked) makes structural analysis challenging

• Dynamic Cell Wall Remodeling: Cell wall composition changes throughout the cell cycle, requiring precise timing of experiments

• Protein Redundancy: Multiple proteins may contribute to β-1,6-glucan synthesis, complicating interpretation of single-protein studies

• Technical Limitations: Difficulties in specifically visualizing β-1,6-glucan without affecting cell wall integrity

• Differential Expression: SPAC17A2.07c is upregulated during septum formation, necessitating synchronized cell populations for certain studies

• Protein Interactions: The target protein likely functions in a complex with other proteins, requiring techniques that preserve these interactions

These challenges necessitate combining antibody-based techniques with genetic, biochemical, and structural approaches for comprehensive analysis.

How can transcriptome analysis complement antibody-based studies of SPAC17A2.07c?

Transcriptome analysis provides valuable complementary data to antibody-based studies of SPAC17A2.07c:

• Global Expression Changes: Analysis of the nmt81-sup11 mutant revealed 439 up-regulated and 239 down-regulated genes, providing context for the protein's function in cellular networks

• Pathway Analysis: Identified significant regulation of several cell wall glucan modifying enzymes, confirming the protein's role in cell wall integrity

• Temporal Dynamics: Can reveal expression patterns throughout the cell cycle or in response to stress

• Genetic Interactions: Identifies genes whose expression changes when SPAC17A2.07c function is compromised

• Novel Targets: May reveal previously unknown players in cell wall synthesis and septum formation

• Validation: Provides orthogonal validation of protein-level findings from antibody-based techniques

Integrating transcriptomic data with antibody-based protein studies offers a more complete understanding of SPAC17A2.07c's role in cellular processes.

How might SPAC17A2.07c research inform antifungal drug development?

Research on SPAC17A2.07c has potential implications for antifungal drug development:

• Novel Drug Targets: As SPAC17A2.07c is essential for cell viability and involved in cell wall formation, it represents a potential target for new antifungal drugs

• Selective Toxicity: The fungal-specific nature of β-1,6-glucan provides an opportunity for drugs that selectively target fungi without affecting human cells

• Resistance Mechanisms: Understanding cell wall synthesis mechanisms could help explain resistance to existing cell wall-targeting antifungals

• Combination Therapies: Insights into cell wall architecture could inform the development of drugs that synergize with existing antifungals

• Cross-Species Applications: Homology with proteins in pathogenic fungi suggests potential broader applications beyond S. pombe

The essential nature of this gene and its specific role in fungal cell biology make it an attractive target for future therapeutic exploration.

What techniques could advance our understanding of SPAC17A2.07c function?

Emerging technologies that could further elucidate SPAC17A2.07c function include:

• CRISPR-Cas9 Gene Editing: For creating precise mutations or conditional alleles to study specific protein domains

• Super-Resolution Microscopy: To visualize the subcellular localization and dynamics of SPAC17A2.07c with nanometer precision

• Proximity Labeling Proteomics: Using techniques like BioID or APEX to identify transient interaction partners in living cells

• Single-Cell Analysis: To understand cell-to-cell variability in SPAC17A2.07c expression and function

• Cryo-Electron Microscopy: For structural determination of SPAC17A2.07c alone or in complex with interaction partners

• Synthetic Genetic Arrays: For systematic identification of genetic interactions, as suggested by search result

• Antibody Engineering: Development of specialized antibodies for specific research applications, drawing on techniques described in search results , , and

Integration of these advanced methodologies would provide unprecedented insights into the function and regulation of SPAC17A2.07c.