SPCC1450.09c Antibody
Description
Introduction to SPCC1450.09c Antibody
SPCC1450.09c Antibody is a research-grade polyclonal antibody developed against the SPCC1450.09c protein from Schizosaccharomyces pombe strain 972 (ATCC 24843), commonly known as fission yeast . This antibody is designed to specifically recognize and bind to the SPCC1450.09c protein, which has been identified in genomic studies of S. pombe. The target protein is referenced in the UniProt database with the accession number Q9Y7N6 .
S. pombe serves as an important model organism in molecular and cellular biology research. It offers advantages such as a relatively simple genome, ease of genetic manipulation, and cellular processes that are often comparable to those in higher eukaryotes. Antibodies against specific S. pombe proteins like SPCC1450.09c are valuable tools for investigating gene function, protein localization, and cellular pathways.
The SPCC1450.09c protein appears to function as a phospholipase, though detailed characterization is still emerging in the research literature . Phospholipases play crucial roles in membrane dynamics, lipid signaling, and cellular metabolism, making them important targets for biological research.
Validated Applications
The SPCC1450.09c Antibody has been validated for specific research applications:
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Enzyme-Linked Immunosorbent Assay (ELISA): The antibody can be used for detecting and quantifying the SPCC1450.09c protein in various sample preparations .
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Western Blotting (WB): This application allows for the identification and semi-quantitative analysis of the target protein in cell or tissue lysates, ensuring proper identification of the antigen .
These applications make the antibody a valuable tool for researchers studying protein expression, regulation, and function in S. pombe. The antibody's specificity for the SPCC1450.09c protein allows for accurate detection in complex biological samples.
Usage Guidelines
For optimal results when working with the SPCC1450.09c Antibody, researchers should follow these guidelines:
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The antibody is designated "For Research Use Only" and should not be used in diagnostic or therapeutic procedures .
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Proper storage at -20°C or -80°C is crucial for maintaining antibody activity. Repeated freeze-thaw cycles should be avoided to prevent degradation of the antibody .
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Appropriate controls should be included in experiments to validate results and ensure specificity of detection.
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Optimization of antibody concentration may be necessary depending on the specific application and sample type.
pombe as a Model Organism
S. pombe serves as an excellent model organism for studying fundamental cellular processes. Research utilizing tools like the SPCC1450.09c Antibody can contribute to our understanding of:
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Membrane biology and lipid metabolism in eukaryotic cells
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Protein function and regulation in cellular pathways
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Evolutionary conservation of biological processes
Fission yeast research has provided valuable insights into cellular mechanisms that are often conserved in higher organisms, making findings potentially relevant to human biology and disease . For example, studies on the TSC pathway in fission yeast have relevance to understanding tuberous sclerosis complex, an autosomal dominant disorder in humans .
Western Blotting Protocol
While specific protocols optimized for the SPCC1450.09c Antibody are not detailed in the available literature, standard Western blotting methods for S. pombe proteins typically include the following steps:
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Sample Preparation: Total cell lysates are prepared by lysing cells with glass beads in appropriate lysis buffer containing protease inhibitors .
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Protein Separation: Equal amounts of total proteins are loaded onto polyacrylamide gels (typically 15% for smaller proteins) and subjected to electrophoresis .
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Transfer: Proteins are transferred to nitrocellulose membranes using standard transfer protocols .
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Antibody Incubation: Membranes are incubated with primary antibodies (such as SPCC1450.09c Antibody) followed by appropriate secondary antibodies. For the SPCC1450.09c Antibody, an anti-rabbit secondary antibody would be suitable .
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Detection: Protein bands are visualized using appropriate detection methods, such as chemiluminescence.
Yeast Culture and Sample Preparation
For studies involving S. pombe and the SPCC1450.09c Antibody, standard yeast culture methods include:
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Growth Conditions: S. pombe cells are typically grown in YEA or EMM medium with appropriate nutrient supplements .
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Cell Lysis: Cells can be lysed using glass beads in lysis buffer containing protease inhibitors such as phenylmethylsulfonyl fluoride and protease inhibitor cocktail .
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Spheroplast Preparation: For certain applications, spheroplasts may be prepared by incubating cells at 37°C in spheroplast buffer .
These general methods provide a foundation for research utilizing the SPCC1450.09c Antibody, though specific optimization may be necessary depending on the particular research objectives.
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
KEGG: spo:SPCC1450.09c
STRING: 4896.SPCC1450.09c.1
Q&A
What is the recommended experimental workflow for validating SPCC1450.09c antibody specificity?
To validate SPCC1450.09c antibody specificity, implement a multi-step process:
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Genetic Controls: Use CRISPR-edited knockout (KO) cell lines of Schizosaccharomyces pombe strain 972/ATCC 24843 as negative controls. Compare immunoblot or immunoprecipitation signals between WT and KO lysates to confirm target recognition .
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Orthogonal Validation: Cross-reference results with RNAi knockdown or alternative detection methods (e.g., mass spectrometry) to exclude off-target binding .
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Dose-Response Testing: Optimize antibody dilution (typically 1:500–1:2000 for Western blotting) to balance sensitivity and background noise .
Example Validation Data Table
| Parameter | WT Signal | KO Signal | Fold Change | Validity Threshold |
|---|---|---|---|---|
| Western Blot Band Intensity | 100% | <5% | >20x | >10x reduction in KO |
| Immunoprecipitation Yield | 1.0 μg | <0.05 μg | >20x | >10x reduction in KO |
How to troubleshoot low signal or high background when using SPCC1450.09c in Western blot?
Optimization Strategies:
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Buffer Composition: Test blocking agents (e.g., 5% BSA vs. non-fat dry milk) to minimize non-specific binding .
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Denaturation Conditions: Adjust SDS-PAGE running time/temperature to preserve epitope integrity.
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Primary Antibody Dilution: Perform titration experiments (e.g., 1:500, 1:1000, 1:2000) to identify optimal concentration .
Critical Controls:
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Include a no-primary-antibody control to assess secondary antibody cross-reactivity.
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Use recombinant SPCC1450.09c protein as a positive control for epitope recognition .
How to integrate SPCC1450.09c into multi-omics studies of S. pombe?
Experimental Design:
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Proteogenomics Correlation: Combine SPCC1450.09c immunoprecipitation with mass spectrometry to identify interacting proteins. Validate hits against RNAi knockdown datasets to exclude false positives .
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Subcellular Localization: Use immunofluorescence (IF) to map SPCC1450.09c localization (e.g., cytoplasmic vs. nuclear). Pair with organelle-specific markers (e.g., mitochondrial HSP70) to resolve spatial dynamics .
Data Integration Example
| Technique | Application | Validation Method |
|---|---|---|
| IP-MS | Protein interaction mapping | KO-based peptide enrichment |
| IF + confocal microscopy | Subcellular localization | Co-staining with organelle markers |
What are the limitations of SPCC1450.09c in cross-species studies?
Key Considerations:
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Species Reactivity: The antibody is validated for S. pombe strain 972/ATCC 24843. Cross-reactivity with Saccharomyces cerevisiae or other fission yeast strains requires empirical testing .
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Epitope Conservation: Sequence alignment of SPCC1450.09c orthologs across species is critical. Low homology in the immunogen region (recombinant S. pombe protein) may limit utility in non-pombe models .
Risk Mitigation:
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Use species-specific knockout models as controls when testing heterologous systems.
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Compare antibody performance with orthogonal detection methods (e.g., qPCR for mRNA) to confirm target engagement .
How to resolve conflicting results when using SPCC1450.09c in different experimental conditions?
Troubleshooting Framework:
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Lot-to-Lot Variability: Compare antibody performance across different batches using KO cell lysates. Recombinant protein spiking can identify batch-specific epitope recognition issues .
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Experimental Context Dependency:
Conflict Resolution Table
| Observed Issue | Potential Cause(s) | Diagnostic Test |
|---|---|---|
| No signal in IP | Insufficient protein expression | qPCR for SPCC1450.09c mRNA |
| Non-specific bands in WB | Cross-reactivity with homologs | KO lysate comparison |
What are best practices for using SPCC1450.09c in immunoprecipitation (IP) workflows?
Protocol Optimization:
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Cell Lysis Conditions: Use mild detergents (e.g., 1% NP-40) to preserve protein-protein interactions. Monitor lysate clarity to avoid aggregation .
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Antibody:Protein Ratio: Perform pull-down efficiency experiments:
Critical Controls:
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IgG Isotype Control: Include non-specific rabbit IgG to exclude bead-binding artifacts.
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Time-Course Experiments: Optimize incubation duration (e.g., 4°C overnight vs. 1 hr room temp) to maximize specificity .
How to interpret Western blot band patterns with SPCC1450.09c?
Data Interpretation Guide:
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Target Band: Expect a single band at the predicted molecular weight (MW) of SPCC1450.09c. Confirm MW via recombinant protein migration .
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Cross-reactive Bands: Smaller/larger bands may indicate:
Example Analysis
| Band Pattern | Interpretation | Action |
|---|---|---|
| Single clean band at MW | Specific binding | Valid result |
| Multiple faint bands | Non-specific binding or degradation | Optimize lysis conditions |
Can SPCC1450.09c be used for functional studies beyond detection?
Functional Assays:
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Antibody-Mediated Inhibition: Test if SPCC1450.09c blocks SPCC1450.09c interaction with binding partners. Validate with RNAi knockdown and rescue experiments .
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ChIP-seq Adaptation: Develop a chromatin immunoprecipitation protocol if SPCC1450.09c is a DNA-binding protein. Requires crosslinking optimization (e.g., formaldehyde concentration) .
Limitations:
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Epitope Accessibility: Conformational epitopes may hinder functional interference. Compare with Fab fragments for improved accessibility .
