SPAC3C7.01c Antibody

What is SPAC3C7.01c and why is it significant in research?

SPAC3C7.01c is a protein-coding gene in Schizosaccharomyces pombe (fission yeast), an organism widely used as a model system in molecular and cellular biology research. Fission yeast serves as an excellent model for studying fundamental cellular processes due to its genetic tractability and similarities to higher eukaryotes. SPAC3C7.01c antibodies enable researchers to study the expression, localization, and function of this protein in various experimental contexts. Fission yeast models like those involving SPAC3C7.01c are particularly valuable for dissecting conserved signaling pathways such as the TSC pathway, which has implications for human diseases .

What applications has the SPAC3C7.01c antibody been validated for?

The SPAC3C7.01c antibody (CSB-PA521098XA01SXV) has been validated for ELISA and Western Blot applications to ensure identification of the target antigen . While these are the validated applications, researchers often optimize and validate antibodies for additional techniques based on their specific experimental needs. The antibody's polyclonal nature may make it suitable for detecting the target protein in various denatured and non-denatured conditions, pending validation by the end-user.

What are the optimal storage conditions for maintaining SPAC3C7.01c antibody activity?

Upon receipt, the SPAC3C7.01c antibody should be stored at -20°C or -80°C to maintain its activity . Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and reduce binding efficiency. The antibody is supplied in liquid form with a storage buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during storage . For working solutions, aliquoting the antibody into single-use volumes is recommended to prevent repeated freezing and thawing of the stock solution.

What are the recommended protocols for using SPAC3C7.01c antibody in Western Blot experiments?

When designing Western Blot experiments with SPAC3C7.01c antibody, researchers should consider the following methodological approach:

• Sample preparation: Extract proteins from S. pombe using established lysis buffers containing protease inhibitors to prevent degradation.

• Protein separation: Separate proteins by SDS-PAGE using an appropriate percentage gel (typically 10-12% for most yeast proteins).

• Transfer and blocking: Transfer proteins to a PVDF or nitrocellulose membrane and block with 5% non-fat milk or BSA in TBST.

• Primary antibody incubation: Dilute SPAC3C7.01c antibody (starting recommendation 1:500 to 1:2000) in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Use an anti-rabbit IgG secondary antibody conjugated with HRP or a fluorescent tag.

• Controls: Include both positive controls (wild-type S. pombe extracts) and negative controls (knockout strains if available) to validate specificity.

Similar to approaches used in antibody characterization studies for other proteins, optimization of antibody dilution and incubation conditions may be necessary to achieve optimal signal-to-noise ratio .

How should researchers design experiments to study SPAC3C7.01c in the context of genetic modifications?

When investigating SPAC3C7.01c in genetically modified contexts, researchers should:

• Generation of mutants: Consider using CRISPR-Cas9 or traditional homologous recombination approaches to create knockouts, point mutations, or tagged versions of SPAC3C7.01c.

• Verification strategy: Use the SPAC3C7.01c antibody in Western Blot to verify the absence of protein in knockout strains or the presence of modified protein in mutant strains.

• Phenotypic analysis: Look for specific phenotypes such as altered growth rates, morphological changes, or responses to environmental stressors, similar to approaches used in fission yeast model studies .

• Complementation tests: Perform genetic complementation by reintroducing wild-type or mutant versions of SPAC3C7.01c to confirm phenotype specificity.

• Pathway analysis: Consider analyzing interactions with known signaling pathways, similar to methods used to study the TSC pathway in fission yeast .

This methodological framework allows for comprehensive analysis of SPAC3C7.01c function within cellular contexts.

What considerations are important when using SPAC3C7.01c antibody in ELISA experiments?

For ELISA applications with SPAC3C7.01c antibody, researchers should consider:

• Antigen preparation: Purified recombinant SPAC3C7.01c protein or cell lysates containing the target protein can be used, depending on the experimental goal.

• Assay format: Direct, indirect, sandwich, or competitive ELISA formats may be employed based on specific research questions.

• Antibody dilution: Start with a dilution series (e.g., 1:500 to 1:10,000) to determine optimal concentration for signal detection.

• Blocking and washes: Use appropriate blocking agents (e.g., BSA, casein) and sufficient washing steps to minimize background.

• Detection system: Select chromogenic, fluorogenic, or chemiluminescent substrates based on required sensitivity.

• Controls: Include positive controls (recombinant SPAC3C7.01c), negative controls (unrelated proteins), and blank wells to validate results.

• Quantification: Develop a standard curve using purified SPAC3C7.01c protein of known concentrations for quantitative analysis.

Similar standardized approaches have proven effective in other antibody characterization studies .

What are common issues when using SPAC3C7.01c antibody and how can they be resolved?

Researchers may encounter several challenges when working with SPAC3C7.01c antibody:

These troubleshooting approaches reflect standardized practices similar to those used in antibody characterization platforms .

How can researchers validate the specificity of SPAC3C7.01c antibody in their experimental system?

Validating antibody specificity is crucial for reliable results. Researchers should:

• Use genetic controls: Compare Western Blot results from wild-type S. pombe strains with SPAC3C7.01c knockout strains, if available. The antibody should detect a band of the expected molecular weight in wild-type samples but not in knockout samples.

• Peptide competition assay: Pre-incubate the antibody with excess purified SPAC3C7.01c protein or immunizing peptide before application. This should eliminate specific binding in Western Blot or immunostaining.

• Immunoprecipitation followed by mass spectrometry: Perform IP with the antibody and analyze the precipitated proteins by mass spectrometry to confirm identity.

• Orthogonal detection methods: Compare protein expression patterns using alternative methods such as GFP-tagged SPAC3C7.01c expressed from its endogenous locus.

• Cross-species reactivity testing: Test the antibody against related proteins from other yeast species to assess cross-reactivity, considering the high specificity for S. pombe strain 972 / ATCC 24843 .

This comprehensive validation approach aligns with best practices established in antibody characterization studies for other targets .

How should researchers interpret conflicting data when using SPAC3C7.01c antibody alongside other detection methods?

When faced with conflicting data between SPAC3C7.01c antibody results and other detection methods:

• Critically evaluate each method: Consider the limitations of each technique. For example, antibody-based methods detect protein levels, while qRT-PCR measures mRNA abundance, which may not always correlate.

• Check for technical issues: Review experimental conditions for each method, including positive and negative controls, to identify potential technical problems.

• Consider protein modifications: Post-translational modifications, protein degradation, or complex formation might affect antibody recognition but not other detection methods.

• Validate with additional approaches: Use orthogonal methods such as mass spectrometry or protein tagging to resolve discrepancies.

• Consult literature: Compare your findings with published data on SPAC3C7.01c or related proteins in fission yeast to provide context for interpretation.

• Design confirmatory experiments: Develop experiments specifically targeted to resolve the conflicting data, such as using multiple antibodies targeting different epitopes.

This analytical approach ensures robust data interpretation and is similar to strategies employed in other protein characterization studies .

How can SPAC3C7.01c antibody be used in studying protein-protein interactions in fission yeast signaling pathways?

For investigating protein-protein interactions involving SPAC3C7.01c:

• Co-immunoprecipitation (Co-IP): Use SPAC3C7.01c antibody to pull down the protein complex from yeast lysates, followed by Western Blot analysis to identify interacting partners. This approach can reveal physiologically relevant interactions within signaling networks.

• Proximity labeling: Combine SPAC3C7.01c antibody with proximity labeling techniques (BioID or APEX) to identify proteins in close proximity to SPAC3C7.01c in living cells.

• Two-hybrid validation: Use antibody detection to validate interactions identified through yeast two-hybrid screens, providing orthogonal confirmation of potential interactors.

• Pathway reconstitution: Apply the antibody to detect SPAC3C7.01c in reconstituted pathway experiments, similar to approaches used in studying the TSC pathway in fission yeast, where protein complexes and their functional interactions are analyzed .

• Cross-linking studies: Combine chemical cross-linking with immunoprecipitation using SPAC3C7.01c antibody to capture transient interactions within signaling cascades.

These advanced applications enable detailed mapping of SPAC3C7.01c's role within cellular signaling networks.

What approaches can researchers use to study SPAC3C7.01c localization and dynamics in living cells?

To investigate SPAC3C7.01c localization and dynamics:

• Immunofluorescence microscopy: Use SPAC3C7.01c antibody in fixed cells with appropriate permeabilization protocols for yeast cell walls to visualize protein localization.

• Fractionation studies: Combine subcellular fractionation with Western Blot using SPAC3C7.01c antibody to biochemically determine protein localization.

• Correlative microscopy: Integrate immunofluorescence with electron microscopy for high-resolution localization studies.

• Dynamic studies: While the antibody itself cannot be used in living cells, researchers can complement antibody studies with live-cell imaging of fluorescently tagged SPAC3C7.01c, validating expression patterns with antibody detection in fixed samples.

• Stress response studies: Monitor changes in SPAC3C7.01c localization under different stress conditions using the antibody, similar to approaches used to study protein dynamics in response to nitrogen starvation in fission yeast .

These methodological approaches provide complementary data on SPAC3C7.01c spatial and temporal regulation.

How can researchers integrate SPAC3C7.01c antibody-based studies with systems biology approaches?

For integrating SPAC3C7.01c studies with systems biology:

• Proteomics integration: Use SPAC3C7.01c antibody for immunoprecipitation followed by mass spectrometry to identify interaction networks, which can be integrated with global proteomic datasets.

• Transcriptomics correlation: Correlate protein expression levels detected by the antibody with transcriptomic data to identify post-transcriptional regulation mechanisms, similar to gene expression profile analysis conducted in fission yeast studies .

• Pathway modeling: Incorporate SPAC3C7.01c antibody-derived data into mathematical models of signaling pathways, particularly in contexts relating to cell growth regulation pathways like those studied in fission yeast .

• Multi-omics data integration: Combine antibody-based protein quantification with metabolomic, transcriptomic, and genomic data to develop comprehensive models of cellular processes involving SPAC3C7.01c.

• Evolutionary studies: Use the antibody to study conservation of SPAC3C7.01c function across yeast species, contributing to evolutionary systems biology perspectives.

This systems-level integration enhances understanding of SPAC3C7.01c's role within the broader cellular context.