SPBC11C11.06c Antibody

What is SPBC11C11.06c and why is it important in fission yeast research?

SPBC11C11.06c is a protein encoded in the genome of Schizosaccharomyces pombe (fission yeast). While limited information is available specifically about SPBC11C11.06c in the search results, it appears to be among the proteins studied in S. pombe research . The importance of studying such proteins lies in understanding fundamental cellular processes in this model organism, which can provide insights into conserved mechanisms across eukaryotes. Antibodies against such proteins enable researchers to detect, localize, and characterize their functions within cellular pathways.

What experimental applications are SPBC11C11.06c antibodies primarily used for?

SPBC11C11.06c antibodies can be used for multiple experimental applications:

• Western blotting for protein detection and quantification

• Immunoprecipitation to study protein complexes and interactions

• Immunofluorescence microscopy for subcellular localization studies

• Protein microarray analysis for specificity testing and cross-reactivity assessment

As demonstrated in studies of other fission yeast proteins, antibodies enable detection of proteins in whole cell extracts, purified cellular fractions, and can be used to track protein expression under varying experimental conditions .

How should researchers evaluate the specificity of a SPBC11C11.06c antibody?

Evaluating antibody specificity is crucial for reliable results. Research shows that antibodies often cross-react with non-target proteins, requiring thorough validation . For SPBC11C11.06c antibody:

• Test against wild-type vs. knockout S. pombe strains

• Perform peptide competition assays

• Validate on whole proteome microarrays containing ~5,000 yeast proteins

• Analyze cross-reactivity patterns through sequence alignment of recognized proteins

• Test under multiple experimental conditions

What are the optimal conditions for using SPBC11C11.06c antibodies in Western blotting?

Based on protocols used for similar yeast protein detection:

• Sample preparation: Use lysis buffer containing:

  • 20 mM HEPES, pH 7.9

  • 100 mM NaCl

  • 1 mM EDTA

  • 10% glycerol

  • Protease inhibitors (0.1 mM Na₃VO₄, 1 mM PMSF, 1 mM DTT)

  • 1% Triton X-100 for cell lysis

• Gel electrophoresis parameters:

  • 6% SDS-polyacrylamide gels for higher molecular weight proteins

  • Loading buffer containing: 100 mM Tris-HCl, pH 7.5, 1.4 M β-mercaptoethanol, 140 mM SDS, 5 mM EDTA, 4 M urea, 1 M thiourea, and 0.72 mM bromophenol blue

• Detection system:

  • Use appropriate primary antibody dilution (typically 1:1000 to 1:5000)

  • HRP-conjugated secondary antibodies

  • ECL reagents for visualization

  • Image capture using systems like ImageQuant LAS 4000

How can I optimize immunoprecipitation protocols for studying SPBC11C11.06c interactions?

For effective immunoprecipitation:

• Cell growth and treatment:

  • Grow cultures to optimal density (OD₆₀₀ of 1.0)

  • Apply relevant treatments (e.g., iron chelators like Dip at 250 μM or FeCl₃ at 100 μM for 1.5-3 hours) to study condition-specific interactions

• Lysis conditions:

  • Use buffer containing detergents suitable for membrane protein solubilization

  • Include protease and phosphatase inhibitors to preserve protein state

  • Consider crosslinking for transient interactions

• Antibody binding:

  • Optimize antibody concentration and incubation time

  • Consider pre-clearing lysates to reduce non-specific binding

  • Use appropriate beads (protein A/G) for antibody capture

• Controls:

  • Include isotype control antibodies

  • Use knockout/knockdown strains as negative controls

  • Validate interactions with reciprocal co-IP where possible

What approaches should I use to visualize SPBC11C11.06c subcellular localization?

For subcellular localization studies, researchers can employ:

• Fluorescence microscopy techniques:

  • Direct immunofluorescence with fluorophore-conjugated primary antibodies

  • Indirect detection using fluorescently-labeled secondary antibodies

  • Co-staining with organelle markers like FM4-64 for vacuoles

• Sample preparation:

  • Optimize fixation methods (paraformaldehyde, methanol)

  • Test different permeabilization conditions

  • Use appropriate blocking agents to minimize background

• Imaging parameters:

  • Utilize multiple channels to detect co-localization

  • Perform z-stack imaging for three-dimensional analysis

  • Consider super-resolution techniques for detailed localization

• Controls and validation:

  • Include GFP-tagged versions of the protein for comparison

  • Use Nomarski optics to examine cell morphology

  • Validate microscopy findings with biochemical fractionation experiments

The study of other S. pombe proteins shows how fluorescence microscopy can be combined with FM4-64 staining to visualize protein localization to specific organelles like vacuole membranes .

How can I use SPBC11C11.06c antibodies to study protein-protein interactions in iron metabolism pathways?

Studies of iron metabolism proteins in S. pombe provide a methodological framework:

• Co-immunoprecipitation strategies:

  • Perform experiments under varying iron conditions (iron-replete vs. iron-depleted)

  • Use iron chelators like 2,2'-dipyridyl (Dip, 250 μM) or iron supplementation (FeCl₃, 100 μM)

  • Analyze interactions with known iron metabolism components

• Experimental design considerations:

  • Include appropriate controls (untreated vs. treated conditions)

  • Consider time-course experiments to capture dynamic interactions

  • Validate interactions with complementary approaches

• Data analysis:

  • Quantify relative abundance of interaction partners

  • Compare interaction profiles under different conditions

  • Integrate findings with known iron metabolism pathways

Research on fission yeast has shown that proteins involved in siderophore biosynthesis (Sib1, Sib2, Sib3) and transport (Str1, Str2) form interaction networks that change under iron-limited conditions .

How can I characterize SPBC11C11.06c expression changes under different stress conditions?

To characterize expression changes:

• Experimental setup:

  • Subject cultures to relevant stress conditions (nutrient limitation, oxidative stress, etc.)

  • Include appropriate time points to capture dynamic changes

  • Prepare whole cell extracts for protein analysis

• Quantitative analysis approaches:

  • Western blotting with normalization to housekeeping proteins (e.g., α-tubulin)

  • Densitometry for semi-quantitative assessment

  • Multiple biological replicates for statistical validity

• Validation strategies:

  • Correlate protein levels with mRNA expression

  • Compare with fluorescently-tagged protein expression

  • Analyze localization changes in addition to expression levels

Studies of other S. pombe proteins demonstrate how expression can be tracked under different conditions, such as iron availability, using immunoblot analysis with appropriate controls .

What strategies can address cross-reactivity issues with SPBC11C11.06c antibodies?

Research has shown that antibody cross-reactivity is a significant challenge, with antibodies recognizing non-target proteins to varying degrees . Strategies to address this include:

• Comprehensive validation:

  • Test against ~5,000 different yeast proteins on proteome microarrays

  • Identify specific cross-reactive proteins

  • Determine if cross-reactivity follows predictable patterns based on sequence homology

• Technical approaches:

  • Antibody affinity purification against specific epitopes

  • Pre-absorption with identified cross-reactive proteins

  • Peptide competition assays to confirm specificity

• Experimental design:

  • Include appropriate genetic controls (knockout/knockdown)

  • Use multiple antibodies targeting different epitopes

  • Compare results with tagged protein versions

How should I interpret conflicting results between antibody-based detection and genetic tagging of SPBC11C11.06c?

When facing discrepancies:

• Systematic analysis:

  • Compare epitope location vs. tag position

  • Evaluate if the tag affects protein function or localization

  • Assess if antibody recognition is affected by post-translational modifications

• Validation experiments:

  • Test under multiple experimental conditions

  • Compare results in different genetic backgrounds

  • Use complementary detection methods

• Resolution approaches:

  • Generate new antibodies targeting different epitopes

  • Create alternative tagged versions with tags in different positions

  • Use genetic complementation to validate functionality

The approach used in fission yeast studies where both antibody detection and GFP-tagging were employed provides a model for resolving such conflicts .

What controls are essential when using SPBC11C11.06c antibodies for immunofluorescence microscopy?

Essential controls include:

• Specificity controls:

  • Genetic controls: SPBC11C11.06c deletion strains

  • Antibody controls: pre-immune serum, isotype controls

  • Staining controls: primary antibody omission, peptide competition

• Localization validation:

  • Co-staining with established organelle markers (e.g., FM4-64 for vacuoles)

  • Comparison with GFP-tagged versions of the protein

  • Cell morphology examination using Nomarski optics

• Technical controls:

  • Fixation and permeabilization controls

  • Autofluorescence assessment

  • Bleed-through controls for multi-channel imaging

Research on S. pombe proteins demonstrates the value of these controls, particularly the use of specific markers for organelles and comparisons between different visualization methods .

How might SPBC11C11.06c antibodies contribute to studying protein function in iron homeostasis pathways?

Based on research in S. pombe iron metabolism:

• Potential research applications:

  • Investigation of SPBC11C11.06c involvement in siderophore biosynthesis pathways

  • Analysis of interactions with known iron transport proteins like Str1 and Str2

  • Characterization of expression and localization under iron limitation

• Methodological approaches:

  • Gene deletion combined with antibody studies of pathway components

  • Growth assays under iron-limited conditions with various supplements

  • Protein-protein interaction studies under varying iron conditions

• Broader implications:

  • Understanding conserved mechanisms of iron homeostasis

  • Insights into stress response pathways in eukaryotic cells

  • Potential applications to fungal pathogenesis research

Studies of fission yeast iron metabolism proteins provide a model for investigating such pathways, revealing complex regulatory networks involving multiple proteins with distinct subcellular localizations .

What emerging technologies could enhance the utility of SPBC11C11.06c antibodies?

Emerging technologies with potential impact include:

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed subcellular localization

  • Live-cell imaging combined with antibody-based detection of fixed timepoints

  • Correlative light and electron microscopy for ultrastructural studies

• Proteomics approaches:

  • Antibody-based proximity labeling (BioID, APEX)

  • Mass spectrometry integration with immunoprecipitation

  • Single-cell proteomics with antibody-based detection

• High-throughput applications:

  • Microfluidic antibody arrays

  • Automated image analysis for large-scale phenotypic studies

  • Integration with CRISPR screens for functional genomics

These emerging tools could significantly enhance our understanding of SPBC11C11.06c function in fission yeast cellular processes.