SPBC18E5.09c Antibody

What is SPBC18E5.09c and what is known about its characteristics?

SPBC18E5.09c is classified as a "sequence orphan" protein in Schizosaccharomyces pombe (fission yeast) . Sequence orphans are proteins with no significant sequence similarity to known proteins in other organisms, making their functional characterization particularly challenging. The gene is located within the genome of S. pombe strain 972 / ATCC 24843 and has been identified during genomic sequencing projects . According to genomic analyses, SPBC18E5.09c is one of the 33 open reading frames (ORFs) identified in the mating-type region of S. pombe, with some of these ORFs containing introns . Despite being sequenced, its specific biological function remains largely uncharacterized.

How are antibodies against sequence orphan proteins like SPBC18E5.09c developed?

Development of antibodies against sequence orphan proteins like SPBC18E5.09c typically follows these methodological steps:

• Antigen design: Computational tools are used to identify immunogenic epitopes specific to the protein sequence.

• Recombinant protein expression: The gene (SPBC18E5.09c) is cloned and expressed, often using systems like E. coli, to produce recombinant protein for immunization.

• Antibody production: Either monoclonal or polyclonal antibodies can be generated:

  • Polyclonal: Multiple epitopes are recognized by immunizing animals (typically rabbits) with the purified protein

  • Monoclonal: Specific hybridoma cell lines are developed to recognize single epitopes

• Purification techniques: Affinity chromatography against the recombinant protein is performed to isolate specific antibodies.

• Validation: The antibody undergoes extensive validation in wild-type and knockout S. pombe strains to confirm specificity .

Commercial providers like Cusabio offer custom SPBC18E5.09c antibodies with specific validation parameters for S. pombe research applications .

What experimental controls should be included when using SPBC18E5.09c antibody?

When designing experiments with SPBC18E5.09c antibody, the following controls are methodologically essential:

• Negative controls:

  • SPBC18E5.09c deletion strain (if viable) to confirm antibody specificity

  • Secondary antibody-only controls to assess non-specific binding

  • Pre-immune serum controls (for polyclonal antibodies)

• Positive controls:

  • Overexpression strains to confirm detection sensitivity

  • Recombinant SPBC18E5.09c protein as a Western blot standard

• Cross-reactivity controls:

  • Testing against related S. pombe proteins to confirm specificity

  • Competition assays with purified antigen

• Procedural controls:

  • Loading controls for Western blots (e.g., α-tubulin)

  • Protocol-specific controls for immunoprecipitation or immunofluorescence

These controls align with standard research protocols outlined for experimental design in molecular biology studies and should be appropriately documented in research protocols .

How can SPBC18E5.09c antibody be used in phosphoproteomic studies?

Integration of SPBC18E5.09c antibody in phosphoproteomic research involves several methodological approaches:

• Immunoprecipitation coupled with mass spectrometry:

  • SPBC18E5.09c antibody can be used to isolate the protein and its interaction partners

  • Subsequent mass spectrometry analysis can identify phosphorylation sites and dynamics

  • This approach has been successfully employed for similar studies in S. pombe as demonstrated in phosphoproteomic networks mediated by Dsk1 kinase

• Phosphorylation-specific antibodies:

  • If phosphorylation sites are identified, phospho-specific antibodies can be developed

  • These can monitor phosphorylation status under different cellular conditions

• Comparative phosphoproteomics:

  • SPBC18E5.09c antibody can be used for enrichment prior to phosphoproteomic analysis

  • Changes in phosphorylation patterns can be monitored using quantitative proteomics approaches like SILAC or TMT labeling

  • This allows for temporal analysis of phosphorylation events during cell cycle or stress responses

• Validation of phosphoproteomics data:

  • Western blotting with SPBC18E5.09c antibody followed by phosphatase treatment can validate mass spectrometry findings

  • Phospho-mutants can be generated and analyzed to determine functional significance

These approaches are similar to those used in characterizing novel targets and phosphoprotein networks in S. pombe cell cycle regulation .

What experimental design considerations are important when studying sequence orphans like SPBC18E5.09c?

When investigating sequence orphans like SPBC18E5.09c, consider these methodological principles:

• Multi-approach functional characterization:

  • Phenotypic analysis of deletion mutants under various stress conditions

  • Localization studies using antibody-based techniques or fluorescent protein tagging

  • Protein-protein interaction mapping through co-immunoprecipitation followed by mass spectrometry

  • Temporal expression analysis during different cell cycle stages and meiosis

• Evolutionary context analysis:

  • Comparative genomic analysis to identify potential orthologs in closely related species

  • Evaluation within the context of ortholog clusters between different organisms, such as those identified between E. coli and S. pombe

• Structured experimental design:

  • Implementation of multiple-probe experimental designs to systematically test hypotheses

  • Temporal staggering of probes to maintain experimental design fidelity

  • Inclusion of remediation steps for potential failures to achieve mastery criteria

• Integration with genomic context:

  • Analysis of the gene's location within the genome (e.g., mating-type region)

  • Evaluation of potential promoter elements and regulatory sequences

  • Assessment of proximity to other genes of known function

• Validation through complementary approaches:

  • Genetic approaches (deletion, overexpression)

  • Biochemical approaches (antibody-based studies)

  • Computational predictions (structural modeling)

A systematic approach combining these methodologies increases the likelihood of assigning functions to orphan sequences .

How can contradictory results with SPBC18E5.09c antibody be resolved?

When faced with contradictory experimental results using SPBC18E5.09c antibody, implement this systematic troubleshooting methodology:

• Antibody validation reassessment:

  • Confirm antibody specificity through Western blotting against wild-type and SPBC18E5.09c deletion strains

  • Test different antibody lots for consistency

  • Consider epitope mapping to confirm target recognition

• Technical parameter optimization:

  • Systematically vary antibody concentrations, incubation times, and buffer compositions

  • Test multiple blocking agents to reduce background

  • Optimize fixation conditions for immunofluorescence applications

• Independent validation approaches:

  • Compare results with tagged versions of SPBC18E5.09c (e.g., GFP or TAP-tagged)

  • Use orthogonal detection methods (mass spectrometry, RNA analysis)

  • Cross-validate with alternative antibodies targeting different epitopes of SPBC18E5.09c

• Biological context considerations:

  • Examine whether contradictions might reflect genuine biological variation (cell cycle state, stress responses)

  • Assess strain background effects by testing in different S. pombe strains

  • Test for post-translational modifications that might affect antibody recognition

• Statistical robustness evaluation:

  • Increase experimental replication to improve statistical power

  • Apply appropriate statistical tests for significance assessment

  • Consider blinded analysis to reduce investigator bias

This structured approach reflects the scientific method applied to antibody-based research problems and aligns with best practices in experimental design .

What are optimal protocols for immunoprecipitation using SPBC18E5.09c antibody?

Optimized immunoprecipitation (IP) with SPBC18E5.09c antibody requires careful methodological consideration:

• Cell lysis optimization:

  • For S. pombe cell wall disruption, use glass bead lysis in buffer containing:

    • 50 mM HEPES, pH 7.5

    • 140 mM NaCl

    • 1 mM EDTA

    • 1% Triton X-100

    • 0.5% sodium deoxycholate

    • Protease inhibitor cocktail

  • Pre-clear lysate by centrifugation (14,000 × g, 15 min, 4°C)

• Antibody binding:

  • Conjugate SPBC18E5.09c antibody to Protein A/G beads or magnetic beads

  • Alternatively, add 2-5 μg antibody to lysate and incubate (4 hours, 4°C) before adding beads

  • For co-immunoprecipitation studies, consider gentler detergent conditions (0.1% NP-40)

• Wash optimization:

  • Perform 4-5 washes with decreasing salt concentrations

  • Include detergent in early washes, remove in later washes

  • For phosphoprotein studies, include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and β-glycerophosphate)

• Elution strategies:

  • Use mild elution with antibody competing peptide for native conditions

  • For denaturing conditions, use SDS sample buffer (95°C, 5 min)

• Controls and validation:

  • Include IgG control IP from same species as SPBC18E5.09c antibody

  • Perform reciprocal IPs when studying protein interactions

  • Validate results with Western blotting of input, unbound, and IP fractions

This protocol incorporates methodologies successfully used in S. pombe protein interaction studies, such as those employed in kinetochore protein analysis .

How can SPBC18E5.09c antibody be used to study protein localization in S. pombe?

For effective subcellular localization studies using SPBC18E5.09c antibody, implement this methodological framework:

• Cell fixation optimization:

  • Test multiple fixation methods:

    • Methanol fixation (-20°C, 6 min) for microtubule preservation

    • 3.7% formaldehyde (25°C, 30 min) for general structure preservation

    • Combined formaldehyde/glutaraldehyde for membrane proteins

  • Optimize spheroplasting conditions for S. pombe cell wall removal

• Immunofluorescence protocol:

  • Permeabilize cells with 1% Triton X-100 in PBS

  • Block with BSA (3%) or normal serum (5-10%)

  • Apply SPBC18E5.09c antibody at optimized concentration (typically 1:100 to 1:1000)

  • Include co-staining with organelle markers for colocalization studies:

    • DAPI for nucleus

    • Tubulin antibody for spindle/microtubules

    • Known centromere markers for kinetochore studies

• Imaging parameters:

  • Collect Z-stack images covering entire cell depth

  • Apply deconvolution algorithms for improved resolution

  • Use quantitative imaging metrics for comparative analysis

• Complementary approaches:

  • Compare immunofluorescence results with live-cell imaging of GFP-tagged SPBC18E5.09c

  • Consider super-resolution microscopy techniques for detailed localization

  • For temporal studies, synchronize cells and examine localization throughout cell cycle

• Validation controls:

  • Include SPBC18E5.09c deletion strains as negative controls

  • Use pre-immune serum controls

  • Validate with second antibody targeting different epitope if available

These approaches have been successfully implemented in studies of S. pombe kinetochore proteins during meiosis and can be adapted for SPBC18E5.09c localization studies .

What approaches can be used to investigate potential functions of the sequence orphan SPBC18E5.09c?

To systematically investigate functions of sequence orphan SPBC18E5.09c, implement this multi-faceted methodology:

• Genetic manipulation approaches:

  • Generate precise gene deletion using CRISPR-Cas9 or homologous recombination

  • Create conditional mutants (temperature-sensitive, auxin-inducible degron)

  • Develop overexpression strains using inducible promoters

  • Perform phenotypic analysis under various conditions (temperature, oxidative stress, DNA damage)

• Transcriptomic analysis:

  • Compare gene expression profiles between wild-type and SPBC18E5.09c deletion strains

  • Identify genes with altered expression using microarray or RNA-seq

  • This approach successfully identified targets of Upf1 in S. pombe

• Protein interaction network mapping:

  • Immunoprecipitation followed by mass spectrometry (IP-MS)

  • Yeast two-hybrid screening

  • Proximity labeling approaches (BioID, APEX)

  • Cross-referencing with known interaction networks, such as those involved in Dsk1 kinase signaling

• Cell cycle analysis:

  • Synchronize cells and assess protein levels/modifications throughout cell cycle

  • Examine potential involvement in specific processes like kinetochore assembly during meiosis

  • Test for genetic interactions with known cell cycle regulators

• Comparative genomics:

  • Search for structural similarities despite absence of sequence conservation

  • Analyze genomic context conservation across related species

  • Investigate potential orthologous relationships as done for E. coli and S. pombe proteins

• Upf1 pathway analysis:

  • Since some sequence orphans are regulated by the Upf1 pathway in S. pombe, investigate if SPBC18E5.09c is a target of nonsense-mediated decay

  • Compare mRNA stability in wild-type versus upf1Δ strains

This comprehensive approach integrates methods successfully applied to characterize other S. pombe genes of initially unknown function and follows established principles of experimental design in molecular biology research .

How should researchers interpret Western blot data using SPBC18E5.09c antibody?

Proper interpretation of Western blot data using SPBC18E5.09c antibody requires this systematic analytical approach:

• Signal specificity assessment:

  • Compare band patterns between wild-type and SPBC18E5.09c deletion strains

  • Expected molecular weight should be calculated from amino acid sequence accounting for potential post-translational modifications

  • Multiple bands may indicate:

    • Degradation products

    • Post-translational modifications

    • Alternative splice variants

    • Cross-reactivity with related proteins

• Quantitative analysis methodology:

  • Normalize target protein signal to loading controls (tubulin, actin)

  • Use image analysis software for densitometry (ImageJ, Image Lab)

  • Apply statistical analysis for comparisons across multiple experiments

  • Express results as fold-change relative to control conditions

• Common interpretation challenges:

  • High background: Optimize blocking conditions, antibody dilution

  • Weak signal: Increase protein loading, enhance detection methods

  • Multiple bands: Validate with tagged versions of the protein

  • Inconsistent results: Standardize lysate preparation and protein quantification

• Advanced analytical considerations:

  • For cell cycle studies, synchronize cultures and collect time points

  • For stress response studies, include positive controls with known response patterns

  • For phosphorylation studies, include phosphatase-treated samples as controls

• Results reporting standards:

  • Include full blot images including molecular weight markers in publications

  • Document all experimental conditions according to research protocol standards

  • Report antibody validation experiments in supplementary materials

This analytical framework aligns with best practices in protein biochemistry and ensures reliable interpretation of Western blot data using SPBC18E5.09c antibody.

What bioinformatic approaches can complement experimental studies with SPBC18E5.09c antibody?

Integrating bioinformatic analysis with experimental data from SPBC18E5.09c antibody studies provides deeper insights through this methodological framework:

• Sequence analysis tools:

  • Secondary structure prediction algorithms (PSIPRED, JPred)

  • Disorder prediction tools (DisEMBL, PONDR)

  • Motif detection algorithms (MEME, ELM)

  • These can identify functional domains despite lack of sequence homology

• Structural prediction approaches:

  • Ab initio protein structure prediction (Rosetta, AlphaFold)

  • Structural comparison with known proteins (DALI server)

  • Molecular dynamics simulations to predict functional sites

• Integrative data analysis:

  • Correlation of proteomic data with transcriptomic profiles

  • Integration with S. pombe genetic interaction networks

  • Mapping to ortholog clusters between E. coli and S. pombe

• Network analysis:

  • Construction of protein-protein interaction networks from IP-MS data

  • Pathway enrichment analysis of interacting partners

  • Network visualization tools (Cytoscape) for identifying functional modules

  • Similar approaches were used to characterize phosphoprotein networks in S. pombe

• Comparative genomics:

  • Synteny analysis across different Schizosaccharomyces species

  • Assessment of selective pressure (dN/dS ratios)

  • Identification of conserved non-coding regulatory elements

• Gene expression correlation analysis:

  • Co-expression pattern analysis with genes of known function

  • Identification of potential regulators and targets

  • Similar to approaches used in characterizing Upf1 targets in S. pombe

These bioinformatic approaches complement experimental data by providing context, suggesting mechanisms, and generating testable hypotheses about SPBC18E5.09c function.

How can researchers establish if SPBC18E5.09c has a role in meiosis or cell cycle regulation?

To systematically investigate SPBC18E5.09c's potential role in meiosis or cell cycle regulation, implement this experimental framework:

• Expression analysis throughout cell cycle and meiosis:

  • Synchronize cells using either:

    • Temperature-sensitive cdc mutants

    • Nitrogen starvation followed by temperature shift in pat1-114 strains

  • Collect samples at defined intervals

  • Quantify protein levels by Western blot with SPBC18E5.09c antibody

  • Compare with established cell cycle markers

• Subcellular localization during meiotic stages:

  • Track protein localization through meiotic prophase, metaphase I/II, and anaphase

  • Co-stain with kinetochore markers to assess potential centromere association

  • Similar approaches revealed dynamic reorganization of kinetochore proteins during meiosis

• Phenotypic characterization of deletion mutants:

  • Measure sporulation efficiency

  • Assess spore viability through tetrad analysis

  • Examine chromosome segregation using techniques like:

    • DAPI staining for nucleus/chromosome visualization

    • Fluorescent protein-tagged histone markers

    • Live-cell imaging of GFP-tagged chromosomal loci

• Genetic interaction analysis:

  • Create double mutants with known meiotic regulators

  • Test interactions with kinetochore components

  • Screen for synthetic phenotypes with cell cycle checkpoint mutants

• Biochemical interaction with cell cycle machinery:

  • Immunoprecipitation with SPBC18E5.09c antibody followed by mass spectrometry

  • Co-immunoprecipitation with known cell cycle regulators

  • Phosphoproteomic analysis to identify cell cycle-dependent modifications

  • Similar approaches revealed phosphoprotein networks in cell cycle regulation

• Functional complementation testing:

  • Replace SPBC18E5.09c with putative functional homologs from related species

  • Test if phenotypes are rescued

This integrated approach has successfully identified roles for previously uncharacterized proteins in S. pombe meiosis and cell cycle regulation .

What information about SPBC18E5.09c antibody validation should be included in research publications?

When publishing research utilizing SPBC18E5.09c antibody, include this comprehensive validation information:

• Antibody specifications:

  • Source (commercial vendor or laboratory-produced)

  • Type (monoclonal or polyclonal)

  • Host species and immunization protocol

  • Lot number for reproducibility

  • Epitope information (peptide sequence or region)

• Validation experiments:

  • Western blot demonstrating specificity (wild-type vs. deletion strains)

  • Immunoprecipitation efficiency assessment

  • Cross-reactivity testing against related proteins

  • Peptide competition assays

  • Side-by-side comparison with epitope-tagged versions of SPBC18E5.09c

• Experimental conditions:

  • Detailed methods section following research protocol guidelines

  • Antibody dilutions/concentrations used

  • Incubation conditions (time, temperature, buffer composition)

  • Detection methods with sensitivity parameters

  • Image acquisition specifications

• Controls documentation:

  • Primary antibody controls (pre-immune serum, isotype controls)

  • Secondary antibody-only controls

  • Loading controls for quantitative comparisons

  • Positive controls (overexpression strains)

  • Negative controls (deletion strains)

• Quantification methodology:

  • Software used for image analysis

  • Statistical methods applied

  • Normalization procedures

  • Number of biological and technical replicates

This comprehensive reporting aligns with emerging standards for antibody validation in research and enhances experimental reproducibility .

How can researchers contribute to the characterization of sequence orphans like SPBC18E5.09c?

Researchers can significantly advance knowledge of sequence orphans like SPBC18E5.09c through these methodological contributions:

• Comprehensive phenotypic characterization:

  • Systematic analysis of deletion phenotypes under diverse conditions

  • Generation of resource strains (tagged versions, conditional alleles)

  • Deposition of strains in community repositories

• Multi-omics data integration:

  • Publication of proteomics, transcriptomics, and genetic interaction data

  • Correlation analysis with existing datasets

  • Network analysis placing the protein in functional contexts

  • Similar to approaches used in characterizing phosphoprotein networks

• Structural characterization:

  • Protein purification protocols optimization

  • Structural determination via X-ray crystallography, cryo-EM, or NMR

  • Computational structure prediction validation

• Community resource development:

  • Generation and validation of research-grade antibodies

  • Development of reporter constructs for functional studies

  • Creation of CRISPR-Cas9 targeting resources

• Data sharing practices:

  • Deposition of datasets in appropriate repositories:

    • Proteomics data in ProteomeXchange

    • RNA-seq data in GEO or ArrayExpress

    • Protein structures in PDB

  • Sharing negative results to prevent duplication of effort

  • Detailed protocols in repositories like protocols.io

• Collaborative approaches:

  • Participation in community annotation projects

  • Contribution to ortholog identification efforts

  • Engagement with computational biology groups for integrated analysis