SPBC1105.08 Antibody

What is SPBC1105.08 and what cellular processes is it involved in?

SPBC1105.08 is a gene in Schizosaccharomyces pombe (fission yeast) that encodes a protein which appears to have roles in cellular regulation. Based on research with related fission yeast proteins, it likely participates in cell cycle regulation, stress response pathways, or cellular structure maintenance . Studies of fission yeast genes often reveal conserved functions relevant to eukaryotic biology more broadly. When designing experiments with the SPBC1105.08 antibody, consider examining protein levels under various growth conditions or stress stimuli to establish baseline expression patterns.

What validation methods should I use to confirm SPBC1105.08 antibody specificity?

To validate SPBC1105.08 antibody specificity, implement multiple complementary approaches:

• Genetic validation: Use SPBC1105.08 knockout strains as negative controls in Western blot and immunofluorescence assays . This approach represents the gold standard for antibody validation.

• Orthogonal validation: Compare protein expression levels between antibody-based assays (e.g., Western blot) and antibody-independent methods (e.g., mass spectrometry) .

• Multiple antibodies approach: Test independent antibodies targeting different epitopes of SPBC1105.08 to confirm consistent results .

• Recombinant expression: Overexpress tagged versions of SPBC1105.08 to confirm detection at expected molecular weight .

Each validation step should be documented with appropriate controls to ensure reproducibility of results.

Which applications is the SPBC1105.08 antibody suitable for?

The SPBC1105.08 antibody (catalog number CSB-PA913562XA01SXV) has been validated for several research applications:

When adapting this antibody to new applications, extensive validation with proper controls is essential for reliable results .

How should I design ChIP-chip experiments using SPBC1105.08 antibody?

When designing ChIP-chip experiments with SPBC1105.08 antibody:

• Sample preparation: Crosslink cells with formaldehyde (typically 1% for 15 minutes) to preserve protein-DNA interactions. Optimize crosslinking time for your specific experiment.

• Chromatin fragmentation: Sonicate to achieve fragments between 200-600bp, verifying size distribution by gel electrophoresis.

• Immunoprecipitation controls: Include IgG controls and input samples. Consider using an antibody concentration of 2-5μg per reaction as a starting point .

• Data analysis: Normalize ChIP-chip data using LOWESS normalization. Define occupancies with enrichment ≥2.5 MAD above array median, similar to approaches used in other fission yeast studies .

• Validation: Confirm key binding sites using targeted ChIP-qPCR.

For example, in similar fission yeast ChIP-chip studies, researchers identified 250 binding sites for transcription factors like Atf1 at probable promoter regions .

What are the best practices for using SPBC1105.08 antibody in co-immunoprecipitation studies?

For co-immunoprecipitation studies with SPBC1105.08 antibody:

• Lysate preparation: Use gentle lysis buffers (e.g., 50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40, with protease/phosphatase inhibitors) to preserve protein-protein interactions.

• Antibody binding: Optimize antibody concentration (typically 1-5μg per sample) and incubation time (4-16 hours at 4°C).

• Bead selection: Use protein A/G beads for most applications; consider using magnetic beads for reduced background.

• Controls: Include:

  • Input samples (5-10% of starting material)

  • IgG control immunoprecipitations

  • Reverse co-IPs to confirm interactions

  • Knockout or knockdown samples when available

• Washing conditions: Balance stringency to remove non-specific interactions while preserving genuine interactions. Start with moderate stringency (e.g., 150mM NaCl) and adjust as needed.

• Elution and detection: Elute proteins with SDS sample buffer and analyze by immunoblotting with antibodies against suspected interaction partners .

How can I use SPBC1105.08 antibody to study protein localization changes during cell cycle progression?

To study SPBC1105.08 localization throughout the cell cycle:

• Synchronization: Synchronize cells using:

  • Temperature-sensitive cdc25 mutants

  • Nitrogen starvation/release

  • Lactose gradient centrifugation

• Sampling: Collect samples at regular intervals (e.g., every 15-20 minutes) covering at least one complete cell cycle.

• Immunofluorescence procedure:

  • Fix cells with 3.7% formaldehyde for 30 minutes

  • Permeabilize cell wall with zymolyase (1mg/ml for 30 minutes)

  • Use nuclear envelope markers like Pom152-GFP to delineate nuclear boundaries

  • Apply SPBC1105.08 antibody (1:100-1:500 dilution range as starting point)

  • Use DAPI staining to visualize DNA

• Imaging: Capture images using deconvolution microscopy to determine subcellular localization patterns.

• Quantification: Measure fluorescence intensity in different cellular compartments across cell cycle stages.

In similar studies with fission yeast proteins, researchers discovered dynamic localization patterns between cytoplasm and nucleus that correlated with specific cell cycle phases . For instance, some proteins like Hst2 were found predominantly in the cytoplasm but also showed nuclear localization during specific cell cycle phases .

What approaches should I use to study SPBC1105.08 interactions with the cell wall in fission yeast?

To investigate potential interactions between SPBC1105.08 and cell wall components:

• Cell wall fractionation: Separate cell wall components through differential extraction:

  • SDS-extraction for loosely associated proteins

  • NaOH-extraction for alkali-sensitive associations

  • Glucanase treatment for covalently linked proteins

• Proteinase K protection assay: Use this approach to determine protein topology relative to the cell wall .

• Cell wall biotinylation: Apply cell-impermeable biotinylation reagents to selectively label cell surface proteins, then immunoprecipitate with SPBC1105.08 antibody and detect with streptavidin .

• Immunoelectron microscopy: Use gold-labeled secondary antibodies to visualize SPBC1105.08 localization at ultrastructural level, particularly in relation to cell wall structures.

• Genetic approaches: Examine interactions with known cell wall synthesis genes through double mutant analysis, similar to studies with sup11+ and β-1,6-glucanase family members .

Research on fission yeast cell wall proteins has identified key interactions between structural components and regulatory proteins that are essential for maintaining cell wall integrity .

How can I use SPBC1105.08 antibody to investigate stress response pathways in fission yeast?

To study the role of SPBC1105.08 in stress response pathways:

• Stress induction protocols:

  • Oxidative stress: Apply H₂O₂ (0.5-1mM)

  • UV irradiation: Standard doses between 10-100 J/m²

  • Nutrient stress: Shift from good to poor nitrogen source

  • Temperature stress: Shift from 30°C to 37°C

• Time-course experiments: Collect samples at multiple time points (0, 15, 30, 60, 120 minutes) after stress induction.

• Protein analysis:

  • Western blot: Detect changes in SPBC1105.08 protein levels

  • Immunoprecipitation: Identify stress-induced interaction partners

  • Phospho-specific detection: Look for post-translational modifications

• ChIP-seq approach: Map SPBC1105.08 genomic binding sites before and after stress, similar to studies of stress-activated MAPK pathways in fission yeast .

• Integration with transcriptome data: Compare SPBC1105.08 binding with gene expression changes during stress response. Consider using SILAC mass spectrometry to map TOR and nutrient-controlled signaling, as done in other fission yeast studies .

In fission yeast, proteins involved in stress response often show altered binding profiles under stress conditions, with significant overlap to CESR (Core Environmental Stress Response) genes .

What are common troubleshooting approaches when SPBC1105.08 antibody produces inconsistent results?

When encountering inconsistent results with SPBC1105.08 antibody:

• Antibody quality control:

  • Verify antibody storage conditions (2-8°C recommended for most antibodies)

  • Check for signs of contamination or precipitation

  • Test a new aliquot or lot of antibody

  • Confirm specificity with appropriate controls

• Sample preparation issues:

  • Ensure complete cell lysis (particularly important for yeast cells)

  • Verify protein extraction efficiency

  • Check for protein degradation by using fresh protease inhibitors

  • Optimize buffer conditions for the particular application

• Technical parameters:

  • Titrate antibody concentration (typically 0.5-5 μg/ml for Western blots)

  • Adjust incubation time and temperature

  • Modify blocking conditions to reduce background

  • Try different detection systems

• Validation experiments:

  • Perform peptide competition assay

  • Test knockout/knockdown samples as negative controls

  • Use recombinant protein as positive control

Approximately 50% of commercial antibodies fail to meet basic standards for characterization, contributing to irreproducible results in research . Thorough validation is essential for reliable experimentation.

How can I distinguish between specific and non-specific signals when using SPBC1105.08 antibody?

To distinguish between specific and non-specific signals:

• Genetic controls: Use SPBC1105.08 knockout or knockdown strains as the most definitive negative control.

• Peptide competition: Pre-incubate antibody with excess immunizing peptide (25-100 μg/ml) to block specific binding sites.

• Cross-adsorption testing: Similar to approaches used for other antibodies, test reactivity against related proteins to assess cross-reactivity .

• Dilution series: Specific signals typically maintain relative intensity across antibody dilutions, while non-specific signals often diminish disproportionately.

• Alternative antibodies: When possible, compare results with independently derived antibodies targeting different epitopes of SPBC1105.08.

• Signal correlation: For microscopy applications, compare localization patterns with known markers or GFP-tagged versions of the protein.

• Size verification: For Western blot applications, confirm that detected bands match theoretical molecular weight of SPBC1105.08.

This systematic approach allows researchers to confidently distinguish between specific and non-specific signals, enhancing experimental reliability .

How can I adapt SPBC1105.08 antibody for single-domain antibody engineering?

For single-domain antibody (sdAb) engineering based on SPBC1105.08 antibody:

• Epitope mapping: Determine the precise binding region using:

  • Peptide arrays

  • Hydrogen-deuterium exchange mass spectrometry

  • X-ray crystallography of antibody-antigen complexes

• CDR identification: Identify complementarity-determining regions (CDRs) within the variable domains of SPBC1105.08 antibody.

• VHH domain isolation: If starting with conventional antibodies, isolate the variable heavy-chain domain as the foundation for sdAb development.

• Recombinant expression systems: Use E. coli, yeast, or mammalian expression systems to produce recombinant sdAbs.

• Affinity maturation: Employ directed evolution approaches (phage display, yeast display) to enhance binding properties.

• Stability optimization: Introduce mutations that enhance thermostability and resistance to aggregation.

• Functional validation: Test engineered sdAbs in the same applications as the parent antibody to confirm maintained specificity and improved properties.

Single-domain antibodies offer advantages including smaller size, better tissue penetration, stability, and cost-effective production compared to conventional antibodies . These properties make them valuable tools for both research and potential therapeutic applications.

What are the best approaches for integrating SPBC1105.08 antibody studies with polysome profiling in fission yeast?

To integrate SPBC1105.08 antibody studies with polysome profiling:

• Synchronized cultures: Start with synchronized cell populations to capture cell cycle-dependent translation events.

• Sample preparation:

  • Treat cells with cycloheximide (100 μg/ml, 5 minutes) to freeze ribosomes on mRNAs

  • Lyse cells gently to preserve polysome integrity

  • Clear lysates by centrifugation (15,000 × g, 10 minutes)

• Sucrose gradient setup:

  • Prepare 7-47% sucrose gradients

  • Layer clarified lysate on gradients

  • Ultracentrifuge (35,000 rpm, 2.5 hours, 4°C)

• Fraction collection:

  • Collect 12-15 fractions while monitoring absorbance at 254 nm

  • Process each fraction for both RNA analysis and protein immunoblotting

• Analysis approaches:

  • RNA: Perform microarray or RNA-seq on fraction RNA to identify transcripts

  • Protein: Perform Western blotting with SPBC1105.08 antibody on each fraction

  • Integration: Correlate SPBC1105.08 protein levels with mRNA translation status

Similar studies in fission yeast have revealed relationships between translation efficiency, mRNA length, and poly(A) tail length . For instance, shorter and more abundant mRNAs typically have longer poly(A) tails and higher translation efficiency .

How can I use SPBC1105.08 antibody to study TOR signaling pathway interactions in fission yeast?

To investigate SPBC1105.08 interactions with TOR signaling:

• Pharmacological manipulation:

  • Treat cells with Torin1 (25 nM) to inhibit TOR signaling

  • Use rapamycin (100 nM) for TORC1-specific inhibition

  • Apply nitrogen stress by changing from good to poor nitrogen source

• Genetic approaches:

  • Use ssp2Δ (AMPK kinase α subunit) strains to eliminate TORC1 inhibition upon nitrogen stress

  • Combine with cdc2 inhibition to eliminate cell cycle fluctuations

• Phosphorylation analysis:

  • Immunoprecipitate SPBC1105.08 using the antibody

  • Perform phospho-specific Western blotting

  • Use SILAC mass spectrometry to identify phosphorylation changes

• Co-immunoprecipitation: Identify TOR pathway components that interact with SPBC1105.08 under different nutritional conditions.

• ChIP experiments: If SPBC1105.08 has DNA-binding properties, examine how TOR inhibition alters its genomic binding profile.

Previous research has shown that TOR signaling responds differently to nitrogen starvation versus nitrogen stress in fission yeast, with distinct phosphoproteome signatures . Over 8,000 phosphorylation sites on 1,920 unique fission yeast proteins were identified in similar studies .

How do SPBC1105.08 antibody-based approaches compare with genetic tagging strategies in fission yeast?

Comparing antibody-based detection with genetic tagging approaches:

For optimal results, researchers should consider combining both approaches:

• Use genetic tagging for initial localization and expression studies

• Validate findings with antibody-based approaches on untagged strains

• Confirm that tagged proteins maintain normal function through complementation tests

In fission yeast studies, researchers have successfully used both approaches to study protein localization. For example, studies of Hst2 localization used myc-tagging combined with immunofluorescence detection .

What methods should I use to optimize SPBC1105.08 antibody performance in different buffer systems?

To optimize SPBC1105.08 antibody performance across buffer systems:

• pH optimization:

  • Test a range of pH values (6.0-8.0, in 0.5 increments)

  • For Western blots, optimize both transfer buffer and blocking/antibody dilution buffers

  • For immunoprecipitation, test binding efficiency across pH range

• Salt concentration:

  • Test NaCl concentrations (50-500mM)

  • Higher salt reduces non-specific binding but may reduce specific signal

  • Optimize separately for binding and washing steps

• Detergent selection:

  • For membrane proteins: Compare Triton X-100, NP-40, digitonin (0.1-1%)

  • For soluble proteins: Test with and without mild detergents

  • Consider specialized detergents for specific applications

• Blocking agents:

  • Compare BSA, milk, casein, and commercial blocking reagents

  • Test concentrations (1-5%)

  • Evaluate blocking time (30 minutes to overnight)

• Additives:

  • Test effects of glycerol (5-10%)

  • Evaluate need for reducing agents (DTT, β-mercaptoethanol)

  • Consider protease/phosphatase inhibitors for sensitive applications

• Systematic optimization:

  • Use a dot blot approach for rapid screening of multiple conditions

  • Develop a scoring system based on signal-to-noise ratio

  • Validate optimal conditions in full experimental setup

Buffer optimization is particularly important for yeast proteins, which may have unique structural features or post-translational modifications affecting antibody recognition.

How can I integrate SPBC1105.08 antibody studies with genome-wide transcriptional profiling in fission yeast?

To integrate SPBC1105.08 antibody studies with transcriptional profiling:

• Experimental design options:

  • Modulate SPBC1105.08 expression (overexpression/knockdown)

  • Create point mutations affecting specific protein domains

  • Apply relevant stress conditions to wild-type and mutant strains

• ChIP-seq approach:

  • Perform chromatin immunoprecipitation with SPBC1105.08 antibody

  • Sequence precipitated DNA to identify genomic binding sites

  • Map binding sites to annotated genome features

• RNA-seq analysis:

  • Extract total RNA from the same experimental conditions

  • Perform RNA-seq to identify differentially expressed genes

  • Compare transcriptional changes with SPBC1105.08 binding sites

• Integration strategies:

  • Identify direct target genes (overlap between binding and expression changes)

  • Perform motif analysis of binding regions

  • Construct gene regulatory networks

• Validation experiments:

  • Confirm key findings with targeted ChIP-qPCR

  • Validate expression changes by RT-qPCR

  • Test functional significance through phenotypic assays

Previous genomic studies in fission yeast have identified 747 cell cycle-regulated genes with expression peaks distributed throughout the cell cycle . Similar approaches could reveal how SPBC1105.08 contributes to gene regulation networks.

What are emerging technologies that could enhance SPBC1105.08 antibody-based research in fission yeast?

Emerging technologies with potential applications for SPBC1105.08 research:

• Proximity labeling techniques:

  • BioID or TurboID fusion proteins to identify neighboring proteins

  • APEX2 for electron microscopy-compatible proximity labeling

  • Integration with mass spectrometry for comprehensive interactome mapping

• Super-resolution microscopy:

  • STORM/PALM for nanoscale localization (20-30nm resolution)

  • Structured illumination microscopy for improved resolution (100nm)

  • Expansion microscopy for physical magnification of structures

• Single-cell approaches:

  • Single-cell Western blotting

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

  • Microfluidic platforms for single-cell analysis

• CRISPR technologies:

  • CUT&RUN or CUT&Tag as alternatives to traditional ChIP

  • CRISPR activation/inhibition to modulate SPBC1105.08 expression

  • Base editing for precise genetic modifications

• Spatial transcriptomics:

  • Correlate SPBC1105.08 localization with spatial gene expression

  • Identify localized translation events related to SPBC1105.08 function

• Computational approaches:

  • Machine learning for image analysis of localization patterns

  • Predictive modeling of protein-protein interactions

  • Network analysis integrating multiple datasets

These technologies could significantly advance understanding of SPBC1105.08 function in fission yeast and potentially reveal conserved mechanisms relevant to other eukaryotic systems.