SPBC8E4.05c Antibody

What is SPBC8E4.05c and why is it important in S. pombe research?

SPBC8E4.05c is an uncharacterized protein from Schizosaccharomyces pombe (fission yeast) that belongs to the fumarate lyase superfamily . The protein is of interest to researchers studying metabolic pathways in yeast, particularly those involving fumarate metabolism. Although its precise function remains to be fully elucidated, its classification within the fumarate lyase superfamily suggests involvement in catalyzing reactions similar to fumarate hydratase or adenylosuccinate lyase. Understanding this protein's role may provide insights into metabolic regulation in eukaryotic systems, as S. pombe serves as an important model organism for studying conserved cellular processes.

What types of SPBC8E4.05c antibodies are available for research?

Currently, researchers can access rabbit polyclonal antibodies against SPBC8E4.05c that are specifically reactive with Schizosaccharomyces pombe strain 972/24843 (Fission yeast) . These antibodies are typically purified using antigen-affinity methods and are of IgG isotype. For researchers requiring the antigen itself for experimental purposes, recombinant SPBC8E4.05c protein is also available with purity levels of 85% or higher as determined by SDS-PAGE analysis . The recombinant protein can be produced in various expression systems including E. coli, yeast, baculovirus, or mammalian cell systems depending on experimental requirements.

What are the validated applications for SPBC8E4.05c antibodies?

SPBC8E4.05c antibodies have been validated for several applications in molecular biology research:

• Western Blotting (WB): For detecting native or denatured SPBC8E4.05c protein in cell lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of the protein

These applications allow researchers to investigate protein expression levels, localization, and potential binding partners. When designing experiments, it's essential to consider that these antibodies have been specifically validated against the strain 972/24843 of S. pombe, which may impact cross-reactivity with other strains or species.

How can I optimize Western blot protocols when using SPBC8E4.05c antibody?

Optimizing Western blot protocols for SPBC8E4.05c detection requires consideration of several technical parameters:

• Sample preparation: Extract proteins from S. pombe cells during logarithmic growth phase to ensure optimal protein expression. Use a lysis buffer containing protease inhibitors to prevent degradation of SPBC8E4.05c protein.

• Gel selection: Given that uncharacterized proteins may have variable migration patterns, initially use gradient gels (4-15%) to determine optimal resolution before switching to fixed percentage gels.

• Antibody dilution: Begin with a 1:500 to 1:1000 dilution for primary antibody incubation, adjusting based on signal strength. For polyclonal antibodies against S. pombe proteins, overnight incubation at 4°C often yields better results than shorter incubations at room temperature.

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBST. If background is high, consider testing both blocking agents, as some antibodies perform better with one versus the other.

• Signal detection: For low abundance proteins like SPBC8E4.05c, enhanced chemiluminescence (ECL) systems with longer exposure times may be necessary. Consider using signal enhancers if detection proves difficult.

Remember that appropriate controls, including positive control lysates from strains known to express SPBC8E4.05c and negative controls from knockout strains, are essential for protocol validation.

What strategies can address cross-reactivity concerns with SPBC8E4.05c antibody?

Cross-reactivity is a significant concern when working with antibodies against proteins from the fumarate lyase superfamily due to conserved structural domains. To address these concerns:

• Pre-absorption: Incubate the antibody with lysates from S. pombe strains with SPBC8E4.05c knocked out to remove antibodies that bind to other proteins.

• Epitope mapping: Determine which regions of SPBC8E4.05c the antibody recognizes and assess sequence homology with other proteins in your experimental system.

• Validation through multiple techniques: Confirm antibody specificity by comparing results from Western blotting with mass spectrometry data or RNA expression analysis.

• Blocking peptide competition: Use a synthetic peptide matching the immunogen to demonstrate specificity by showing that pre-incubation with this peptide abolishes the specific signal.

• Genetic validation: If possible, use genetic approaches such as tagged versions of SPBC8E4.05c or gene knockout/knockdown studies to verify antibody specificity.

Cross-reactivity assessment is particularly important when studying protein families with high sequence or structural similarity, as is often the case with metabolic enzymes like those in the fumarate lyase superfamily.

How can I integrate SPBC8E4.05c antibody studies with genetic manipulation in S. pombe?

Integrating antibody-based detection with genetic manipulation provides powerful insights into protein function. For SPBC8E4.05c research:

• CRISPR/Cas9 modification: Generate knockout or tagged versions of SPBC8E4.05c to validate antibody specificity and study protein function. When designing guide RNAs, avoid regions corresponding to antibody epitopes if you plan to use the antibody with tagged constructs.

• Mating-type switching systems: Leverage S. pombe's well-characterized mating-type switching system to study SPBC8E4.05c expression in different cell types. The Swi2-Swi5 complex, which is central to mating-type determination, can provide a model system for studying regulatory mechanisms .

• Complementary approaches: Combine antibody detection with fluorescent protein tagging to correlate results from fixed-cell immunostaining with live-cell imaging.

• Inducible expression systems: Use nmt1 promoter-based expression systems with varying thiamine concentrations to study the effects of SPBC8E4.05c overexpression, followed by antibody-based detection to confirm expression levels.

• Epistasis analysis: Use the antibody to detect protein levels in various genetic backgrounds to place SPBC8E4.05c in a functional pathway.

This integrated approach allows for robust validation of antibody specificity while generating functional insights about the protein of interest.

What controls should be included when performing immunoprecipitation with SPBC8E4.05c antibody?

Immunoprecipitation (IP) experiments with SPBC8E4.05c antibody require rigorous controls to ensure reliable results:

When analyzing IP results, compare band patterns across all controls to identify truly specific interactions. For co-immunoprecipitation studies investigating protein-protein interactions, additional controls may be necessary to rule out RNA-mediated interactions, particularly when working with nucleic acid-binding proteins.

How do I interpret conflicting results between antibody-based detection and transcriptomic data?

Discrepancies between protein detection using SPBC8E4.05c antibody and mRNA expression data are not uncommon and can provide valuable biological insights:

• Post-transcriptional regulation: Investigate whether SPBC8E4.05c is subject to regulatory mechanisms such as microRNA targeting, RNA binding protein regulation, or altered mRNA stability. Test for these possibilities by measuring mRNA half-life or performing RNA-protein interaction studies.

• Post-translational modifications: The antibody may have differential affinity for modified forms of the protein. Utilize phosphatase treatments or specific modification-detecting antibodies to determine if this explains the discrepancy.

• Protein stability differences: Measure protein half-life using cycloheximide chase experiments to determine if the protein has unexpectedly long or short stability.

• Technical considerations: Verify antibody specificity using knockout controls and consider whether sample preparation methods preserve the epitope recognized by the antibody.

• Biological compartmentalization: The protein may be sequestered in cellular compartments that are differentially extracted during sample preparation. Perform fractionation experiments to test this hypothesis.

These investigations can transform an apparent experimental inconsistency into a novel discovery about SPBC8E4.05c regulation.

What approaches can verify SPBC8E4.05c antibody specificity in structural studies?

Verifying antibody specificity is critical for structural biology applications:

• Epitope mapping: Determine the specific amino acid sequence recognized by the antibody using peptide arrays or hydrogen-deuterium exchange mass spectrometry (HDX-MS). This information can be cross-referenced with structural databases like SAbDab to predict antibody binding sites .

• Competitive binding assays: Use defined fragments of SPBC8E4.05c to compete for antibody binding, confirming the structural requirements for recognition.

• Cross-linking coupled with mass spectrometry: Identify the precise antibody-antigen interaction sites by cross-linking the complex and analyzing the linked peptides by mass spectrometry.

• Cryo-EM validation: For structural studies utilizing antibodies as fiducial markers, validate binding specificity by comparing structures with and without antibody labeling.

• Comparative analysis with canonical classifications: For antibodies targeting specific structural motifs, compare their binding patterns with established structural classifications from databases like SAbDab .

These approaches provide structural validation of antibody specificity beyond functional assays, which is particularly important when using antibodies as structural probes.

How can SPBC8E4.05c antibody be used to investigate protein interactions within the fumarate lyase superfamily?

Investigating protein interactions within enzyme families requires specialized approaches:

• Co-immunoprecipitation with metabolic complex isolation: Use SPBC8E4.05c antibody to pull down protein complexes under native conditions, followed by mass spectrometry to identify interacting partners. Compare results obtained during different metabolic states to detect condition-specific interactions.

• Proximity labeling: Combine SPBC8E4.05c antibody detection with BioID or APEX2 proximity labeling to identify proteins that are proximal to SPBC8E4.05c in living cells, providing spatial context for potential interactions.

• Competitive binding studies: Use purified recombinant proteins from the fumarate lyase superfamily to determine if they compete for the same binding partners identified in co-IP experiments.

• Substrate channeling investigation: If SPBC8E4.05c functions in a metabolic pathway, use the antibody to detect whether it forms complexes with enzymes that act upstream or downstream in the pathway, potentially facilitating substrate channeling.

• In situ visualization: Perform immunofluorescence using SPBC8E4.05c antibody alongside antibodies against other fumarate lyase superfamily members to determine co-localization patterns within cells.

These approaches can reveal whether SPBC8E4.05c functions independently or as part of larger metabolic complexes, providing insights into metabolic organization in S. pombe.

What are effective methods for quantifying SPBC8E4.05c expression changes in response to metabolic stress?

Quantifying protein expression changes during stress responses requires careful experimental design:

• Quantitative Western blotting: Use SPBC8E4.05c antibody in Western blots with internal loading controls (such as tubulin or GAPDH) and standard curves generated with recombinant protein to accurately quantify expression changes.

• ELISA development: Establish a sandwich ELISA using SPBC8E4.05c antibody for high-throughput quantification across multiple samples . This approach is particularly valuable for time-course experiments examining adaptation to stress conditions.

• Flow cytometry: For single-cell analysis of expression heterogeneity, optimize intracellular staining protocols using SPBC8E4.05c antibody for flow cytometry.

• Multiplexed detection: Combine SPBC8E4.05c antibody with antibodies against stress response proteins in multiplexed assays (such as Luminex or antibody arrays) to correlate SPBC8E4.05c expression with broader stress response patterns.

• Time-resolved analysis: Implement pulse-chase labeling combined with immunoprecipitation to distinguish between changes in protein synthesis versus degradation rates during stress adaptation.

When designing these experiments, ensure appropriate statistical approaches for analyzing quantitative changes, including sufficient biological and technical replicates to account for natural variation in stress responses.

How can SPBC8E4.05c antibody be integrated with mating-type studies in S. pombe?

S. pombe's mating-type switching system offers a unique context for studying protein expression:

This approach leverages S. pombe's well-characterized sexual differentiation system to provide context for understanding SPBC8E4.05c function.

What strategies can address weak or inconsistent signals when using SPBC8E4.05c antibody?

When encountering weak or variable signals, consider these methodological adjustments:

• Sample preparation optimization:

  • Modify lysis buffers to improve protein extraction efficiency

  • Test different detergents (CHAPS, NP-40, Triton X-100) for optimal solubilization

  • Include protease and phosphatase inhibitors to prevent degradation or modification

  • Standardize cell growth conditions to minimize biological variability

• Antibody optimization:

  • Test different antibody concentrations ranging from 1:100 to 1:2000

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try different blocking agents (BSA, casein, commercial blocking solutions)

  • Consider using signal amplification systems such as biotin-streptavidin

• Technical considerations:

  • Ensure transfer efficiency for Western blots by using stain-free gels or Ponceau staining

  • Optimize antigen retrieval methods for immunohistochemistry or fixed-cell immunofluorescence

  • Verify that the epitope isn't masked by sample preparation methods

  • Consider native versus denaturing conditions if conformational epitopes are involved

Systematic optimization of these parameters while maintaining appropriate experimental controls can significantly improve signal quality and consistency.

How do experimental conditions affect SPBC8E4.05c antibody binding characteristics?

Different experimental conditions can dramatically impact antibody performance:

When optimizing these conditions, change only one parameter at a time while keeping others constant to effectively identify critical factors affecting antibody performance in your specific experimental system.