SPCC663.13c Antibody

What is SPCC663.13c and why is it important in fission yeast research?

SPCC663.13c is a protein encoded in the genome of Schizosaccharomyces pombe (fission yeast), with UniProt accession number O74519 . While not extensively characterized in the provided literature, it belongs to a chromosomal region that includes several genes involved in cellular stress responses. The protein is significant in S. pombe research as it may participate in oxidative stress response pathways, similar to other proteins in this region such as SPCC663.08c which has been identified in studies examining oxidative stress mechanisms . Understanding SPCC663.13c function contributes to our knowledge of cellular redox homeostasis in this model organism, which has significant evolutionary conservation with higher eukaryotes including humans.

What are the key characteristics of the SPCC663.13c Antibody?

The SPCC663.13c Antibody (Product Code: CSB-PA527935XA01SXV) is a polyclonal antibody raised in rabbits against a recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) SPCC663.13c protein . The antibody has the following characteristics:

• Clonality: Polyclonal

• Host: Rabbit

• Isotype: IgG

• Form: Liquid

• Conjugation: Non-conjugated

• Purification method: Antigen affinity purified

• Storage buffer: 0.03% Proclin 300 as preservative, 50% Glycerol, 0.01M PBS, pH 7.4

These characteristics make it suitable for detecting the native SPCC663.13c protein in various research applications focused on S. pombe cellular biology.

What experimental applications is the SPCC663.13c Antibody validated for?

The SPCC663.13c Antibody has been validated for the following applications:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

These applications enable researchers to detect and quantify SPCC663.13c protein in complex biological samples. While not explicitly validated for other techniques, similar polyclonal antibodies may potentially be useful for immunoprecipitation or immunofluorescence studies, though this would require additional validation by researchers.

What is the recommended storage protocol for maintaining SPCC663.13c Antibody activity?

To maintain optimal activity of the SPCC663.13c Antibody, the manufacturer recommends:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles as these can denature antibodies and reduce their effectiveness

• The antibody is supplied in 50% glycerol buffer which helps prevent freezing damage

For working solutions, aliquoting the stock antibody into smaller volumes before freezing is advisable to minimize freeze-thaw cycles. Short-term storage (1-2 weeks) at 4°C is possible for working dilutions, but longer storage should be at recommended freezing temperatures.

How does SPCC663.13c relate to oxidative stress response pathways in S. pombe?

While specific information about SPCC663.13c's role in oxidative stress response is limited in the provided search results, we can infer potential connections based on related proteins. In S. pombe, the genomic region containing SPCC663 genes appears to include several stress response components. For example, SPCC663.08c was identified in studies examining oxidative stress mechanisms and was shown to be transcriptionally regulated in response to hydrogen peroxide treatment .

The study by García-Santamarina et al. demonstrated that several proteins in this genomic region show differential expression patterns in strains lacking thioredoxin (Δtrx1) or thioredoxin reductase (Δtrr1) . These proteins often contain redox-sensitive cysteine residues that undergo reversible oxidation. Analysis of the Northern blot data indicates that SPCC663.08c shows increased transcription in response to H₂O₂ treatment, suggesting a potential role in the cellular response to oxidative stress .

Based on its genomic proximity and potential functional relationships, SPCC663.13c may similarly participate in redox homeostasis pathways, making the antibody valuable for researchers investigating oxidative stress responses in fission yeast.

What considerations should researchers make when designing experiments with SPCC663.13c Antibody?

When designing experiments with SPCC663.13c Antibody, researchers should consider:

• Strain selection: The antibody has been designed specifically for Schizosaccharomyces pombe strain 972/ATCC 24843 . Using different strains may affect epitope recognition and antibody performance.

• Expression levels: Consider that expression of stress-response proteins may vary significantly under different growth conditions or stress treatments. Based on similar proteins in the SPCC663 region, expression might be induced by oxidative stress conditions such as H₂O₂ treatment .

• Protein modifications: If SPCC663.13c contains redox-sensitive cysteines, its detection may be affected by sample preparation methods that alter the redox state. García-Santamarina et al. used specialized techniques like isotope-coded affinity tag (ICAT) methodology to identify reversibly oxidized peptides .

• Controls: Include appropriate positive and negative controls. For S. pombe proteins, this might include:

  • Wild-type strain 972 (positive control)

  • Knockout strains (negative control)

  • Oxidative stress-induced and non-induced samples for comparison

• Antibody specificity: Validate specificity through appropriate controls such as pre-adsorption with the immunizing peptide or parallel analysis with knockout strains.

How can researchers validate the specificity of SPCC663.13c Antibody?

Validating antibody specificity is crucial for reliable experimental results. For SPCC663.13c Antibody, researchers should consider these validation approaches:

• Genetic validation: Compare antibody reactivity between wild-type S. pombe and a SPCC663.13c deletion strain. Absence of signal in the deletion strain would strongly support antibody specificity.

• Overexpression validation: Test the antibody against samples overexpressing tagged SPCC663.13c protein. Following approaches similar to those used by García-Santamarina et al. for other S. pombe proteins, researchers could generate strains expressing HA-tagged SPCC663.13c and compare detection with both anti-HA and the SPCC663.13c antibody .

• Mass spectrometry correlation: Following immunoprecipitation with the SPCC663.13c antibody, analyze the precipitated proteins by mass spectrometry to confirm the presence of SPCC663.13c peptides.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application to samples. Specific binding should be significantly reduced or eliminated.

• Cross-reactivity assessment: Test the antibody against related proteins or in other species to assess potential cross-reactivity that might complicate data interpretation.

What is the recommended protocol for using SPCC663.13c Antibody in Western blotting?

While specific manufacturer's protocols for SPCC663.13c Antibody are not detailed in the search results, a standard Western blotting protocol for S. pombe proteins can be adapted based on methodologies described by García-Santamarina et al. for similar proteins :

• Sample preparation:

  • Harvest S. pombe cells (typically 10-50 ml of culture at OD600 ~0.5)

  • Extract proteins using trichloroacetic acid (TCA) precipitation:

    • Add TCA to a final concentration of 10-20%

    • Incubate on ice for 15 minutes

    • Centrifuge and wash precipitate with acetone

    • Resuspend in appropriate sample buffer

• Gel electrophoresis:

  • Separate proteins using SDS-PAGE (typically 10-12% polyacrylamide)

  • Include molecular weight markers

• Transfer:

  • Transfer proteins to PVDF or nitrocellulose membrane

• Blocking:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute SPCC663.13c Antibody (recommended starting dilution: 1:1000, though optimization may be required)

  • Incubate membrane overnight at 4°C

• Washing:

  • Wash membrane 3-4 times with TBST, 5-10 minutes each

• Secondary antibody incubation:

  • Use anti-rabbit IgG conjugated to HRP (typically 1:5000 dilution)

  • Incubate for 1 hour at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL) reagent

  • Expose to X-ray film or image using a digital imaging system

How should sample preparation be optimized when working with SPCC663.13c Antibody in ELISA?

For optimal ELISA results with SPCC663.13c Antibody, consider these sample preparation guidelines:

• Cell lysis optimization:

  • For S. pombe, use glass bead lysis in a buffer containing:

    • 50 mM Tris-HCl, pH 7.5

    • 150 mM NaCl

    • 0.1% Triton X-100

    • Protease inhibitor cocktail

  • Alternatively, TCA extraction provides good preservation of protein modifications

• Protein quantification:

  • Perform Bradford or BCA assay to ensure equal loading across samples

  • Standardize protein concentration to 1-5 μg/ml for coating ELISA plates

• Preservation of protein state:

  • If studying redox state, include alkylating agents in lysis buffer to preserve cysteine oxidation states

  • Based on protocols for similar proteins, consider using ICAT (isotope-coded affinity tag) methodology for studying cysteine oxidation

• Sample handling:

  • Minimize freeze-thaw cycles

  • Process samples promptly after collection

  • Consider adding phosphatase inhibitors if phosphorylation may affect epitope recognition

• Detergent considerations:

  • Ensure detergent concentrations are compatible with the ELISA format

  • For membrane-associated proteins, gentle detergents like 0.1% Triton X-100 or 0.1% NP-40 may improve extraction

What controls should be included when using SPCC663.13c Antibody in experimental procedures?

To ensure experimental rigor when using SPCC663.13c Antibody, include these essential controls:

• Positive controls:

  • Wild-type S. pombe strain 972 (the antibody's target species and strain)

  • If available, recombinant SPCC663.13c protein used as the immunogen

  • Samples with known or induced expression of SPCC663.13c

• Negative controls:

  • SPCC663.13c deletion strains

  • Non-target species extracts

  • Primary antibody omission control

  • Isotype control (non-specific rabbit IgG at the same concentration)

• Validation controls:

  • Peptide competition assay to confirm specificity

  • Gradient of protein concentrations to establish linearity of signal

• Technical controls:

  • Loading controls for Western blots (e.g., anti-tubulin or anti-actin)

  • Standard curves for quantitative applications

• Experimental condition controls:

  • For oxidative stress studies, include both treated (e.g., H₂O₂) and untreated samples

  • When studying protein-protein interactions, include controls for non-specific binding

Following the example of García-Santamarina et al., researchers might consider generating tagged versions of SPCC663.13c (e.g., HA-tagged) to enable parallel detection with both anti-HA and SPCC663.13c antibodies as an additional validation approach .

How can researchers troubleshoot common issues with SPCC663.13c Antibody in Western blotting?

When encountering problems with SPCC663.13c Antibody in Western blotting, consider these troubleshooting approaches:

For S. pombe proteins specifically, consider that expression may be condition-dependent, as seen with oxidative stress-responsive proteins in the study by García-Santamarina et al.

What are the key considerations for detecting native versus denatured SPCC663.13c protein?

When planning experiments to detect SPCC663.13c in different conformational states, researchers should consider:

• Native conditions:

  • For detecting proteins in their native conformation (e.g., for co-immunoprecipitation):

    • Use gentler lysis methods (avoid TCA precipitation)

    • Maintain protein-protein interactions with buffers containing:

      • 25-50 mM Tris-HCl pH 7.5

      • 100-150 mM NaCl

      • 0.1-0.5% NP-40 or Triton X-100

      • Protease inhibitor cocktail

    • Avoid harsh detergents, reducing agents, and extreme pH

• Denatured conditions:

  • For Western blotting and other applications requiring denatured protein:

    • Use stronger lysis methods (SDS, urea, or TCA precipitation)

    • Add reducing agents like β-mercaptoethanol or DTT if epitope recognition requires reduced cysteines

    • Apply heat denaturation (95°C for 5-10 minutes)

• Redox considerations:

  • If SPCC663.13c contains redox-sensitive cysteines (like many stress-response proteins):

    • For studying oxidized forms: avoid reducing agents and alkylate free thiols during extraction

    • For studying reduced forms: include reducing agents like DTT or TCEP

    • Consider specialized techniques like ICAT labeling as used by García-Santamarina et al.

• Epitope accessibility:

  • The polyclonal nature of this antibody may provide recognition of multiple epitopes, some of which may be accessible only in certain conformations

  • Consider whether the recombinant immunogen used to generate the antibody was in native or denatured form

How can the SPCC663.13c Antibody be used in studies examining oxidative stress responses?

Based on research methodologies for related proteins in S. pombe, the SPCC663.13c Antibody could be valuable in oxidative stress research using these approaches:

• Expression profiling:

  • Compare SPCC663.13c protein levels between:

    • Wild-type and oxidative stress mutants (e.g., Δtrx1, Δtrr1)

    • Untreated cells versus H₂O₂-treated cells

    • Different time points during stress response

• Redox state analysis:

  • Adapt the ICAT methodology used by García-Santamarina et al. to examine cysteine oxidation in SPCC663.13c :

    • Label reduced cysteines with light ICAT reagent

    • Label oxidized cysteines (after reduction) with heavy ICAT reagent

    • Analyze by mass spectrometry to determine oxidation ratio

  • Alternatively, use mobility shift assays with and without reducing agents

• Protein interaction studies:

  • Use co-immunoprecipitation with SPCC663.13c Antibody to identify protein partners

  • Compare interaction profiles under normal versus oxidative stress conditions

  • Consider using thioredoxin mutants like Trx1.C33S for trapping mixed disulfides if SPCC663.13c is a thioredoxin substrate

• Subcellular localization:

  • If validating the antibody for immunofluorescence microscopy:

    • Compare localization under normal versus oxidative stress conditions

    • Co-localize with known stress response proteins

• Functional studies:

  • Create SPCC663.13c knockout or overexpression strains

  • Use the antibody to verify deletion or overexpression

  • Examine phenotypes under normal and stressed conditions

This methodological approach parallels the comprehensive study of thioredoxin-dependent proteins by García-Santamarina et al., providing a framework for investigating SPCC663.13c's potential role in oxidative stress responses .

How might SPCC663.13c Antibody contribute to understanding redox regulation in eukaryotic cells?

The SPCC663.13c Antibody represents a valuable tool for expanding our understanding of redox regulation in eukaryotic cells, with several promising research applications:

• Mapping the oxidative stress response network:

  • S. pombe serves as an excellent model for eukaryotic stress responses

  • SPCC663.13c may be part of evolutionarily conserved redox pathways

  • The antibody enables identification of this protein's role within the network

  • It could reveal connections to known redox proteins such as thioredoxin, similar to other proteins identified in García-Santamarina et al.'s study

• Identifying post-translational modifications:

  • Redox-sensitive proteins often undergo complex modifications

  • The antibody can be used to immunoprecipitate SPCC663.13c for subsequent analysis of:

    • Cysteine oxidation states

    • Phosphorylation events triggered by stress

    • Other modifications that regulate activity

• Comparative studies across species:

  • If homologs exist in other organisms, cross-reactivity testing could reveal conservation

  • This would contribute to understanding fundamental redox mechanisms across evolution

• Integration with -omics approaches:

  • Combining antibody-based detection with techniques from García-Santamarina et al. such as:

    • ICAT labeling for redoxomics

    • Proteomics to identify interaction partners

    • Transcriptomics to correlate protein levels with gene expression

These applications position the SPCC663.13c Antibody as an important reagent for expanding our understanding of fundamental cellular processes related to oxidative stress management.

What are potential emerging applications for SPCC663.13c Antibody in systems biology approaches?

As systems biology continues to evolve, the SPCC663.13c Antibody could find application in several cutting-edge methodologies:

• Integrated multi-omics studies:

  • Coupling antibody-based detection with:

    • Transcriptomics (RNA-seq)

    • Proteomics (mass spectrometry)

    • Metabolomics

  • This would position SPCC663.13c within global cellular networks

• Single-cell analysis:

  • Adaptation of the antibody for flow cytometry or microfluidics-based single-cell analysis

  • This could reveal cell-to-cell variability in SPCC663.13c expression and localization during stress responses

• Temporal dynamics studies:

  • Using the antibody to track SPCC663.13c levels and modifications:

    • During acute stress response

    • Through adaptation phases

    • In recovery periods

  • This temporal resolution would provide insights into the dynamics of stress management

• Synthetic biology applications:

  • Characterizing SPCC663.13c for potential incorporation into engineered stress response circuits

  • The antibody would enable validation and monitoring of synthetic systems

• Computational modeling validation:

  • Using quantitative data from antibody-based assays to:

    • Validate in silico models of stress response pathways

    • Refine parameters for systems biology simulations

    • Test predictions about network behavior

These emerging applications highlight the potential value of SPCC663.13c Antibody beyond traditional biochemical approaches, positioning it as a tool for cutting-edge integrative biology research.

How does current research on S. pombe stress responses inform the study of SPCC663.13c?

Current research on S. pombe stress responses provides important context for studying SPCC663.13c:

• Thioredoxin system connections:

  • García-Santamarina et al.'s research revealed that many proteins show altered oxidation states in thioredoxin-deficient (Δtrx1) or thioredoxin reductase-deficient (Δtrr1) strains

  • If SPCC663.13c functions in related pathways, it may show similar regulatory patterns

  • The SPCC663.13c Antibody would enable testing of this hypothesis through comparative studies with wild-type and mutant strains

• Transcription factor regulation:

  • Research has identified several transcription factors controlling stress responses in S. pombe, including Pap1

  • The antibody could help determine if SPCC663.13c is regulated by these transcription factors by comparing protein levels in transcription factor mutants

• Metabolic adaptation:

  • S. pombe undergoes significant metabolic remodeling during oxidative stress

  • If SPCC663.13c participates in these pathways, the antibody would enable correlation studies between protein expression and metabolic changes

• Conservation with human systems:

  • Many S. pombe stress response mechanisms are conserved in humans

  • Understanding SPCC663.13c's role using the antibody could provide insights relevant to human health and disease, particularly in conditions involving oxidative stress

This research context provides a framework for using the SPCC663.13c Antibody to address specific hypotheses about this protein's function within established stress response networks in S. pombe.