SPBC1198.03c Antibody

What is SPBC1198.03c and what is its significance in fission yeast research?

SPBC1198.03c is an uncharacterized protein in Schizosaccharomyces pombe (fission yeast). Although its precise function remains to be fully elucidated, genome-wide deletion studies have identified it as a non-essential gene (Hayles et al., 2013) . The protein shows specific subcellular localization patterns, being present in the cytoplasm and nucleus, with particular concentration at the cell tip and barrier septum.

This localization pattern suggests potential roles in cell division, polarity maintenance, or cell wall formation. For researchers using S. pombe as a model organism, studying SPBC1198.03c contributes to our understanding of conserved eukaryotic cellular processes, particularly those related to cell cycle regulation and chromosome dynamics.

What are the validated applications for SPBC1198.03c antibody?

Based on product characterization data, SPBC1198.03c antibody has been validated for the following applications:

The antibody is a polyclonal IgG raised in rabbits against recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) SPBC1198.03c protein . It has been purified using antigen affinity chromatography to enhance specificity. While these are the confirmed applications, researchers may need to optimize conditions for their specific experimental systems.

How should SPBC1198.03c antibody be stored and handled for optimal performance?

For optimal preservation of antibody activity and specificity, the following storage and handling protocols are recommended:

• Upon receipt, store at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles that can degrade antibody performance

• The antibody is supplied in liquid form with a storage buffer containing 0.03% Proclin 300 (preservative), 50% Glycerol, and 0.01M PBS, pH 7.4

• For routine use, small aliquots can be prepared to minimize freeze-thaw cycles

• When handling, maintain cold chain whenever possible and use sterile technique to prevent contamination

• Before each use, centrifuge the antibody vial briefly to collect solution at the bottom

What validation methods ensure SPBC1198.03c antibody specificity?

While specific validation data for commercial SPBC1198.03c antibodies varies between manufacturers, standard validation methodologies typically include:

• Antigen-specific validation: Testing against recombinant SPBC1198.03c protein to confirm binding specificity

• Application testing: Validation in specific applications such as ELISA and Western blot to ensure performance in those experimental contexts

• Purification method: Antigen affinity purification to select antibodies with high specificity for the target

• Species reactivity assessment: Confirmation of reactivity with Schizosaccharomyces pombe (strain 972/ATCC 24843) proteins

Researchers should note that additional validation may be required for applications beyond those specifically tested by manufacturers, particularly for specialized techniques like ChIP or immunofluorescence.

How can SPBC1198.03c antibody be optimized for chromatin immunoprecipitation (ChIP) experiments?

While SPBC1198.03c antibody has not been specifically validated for ChIP applications in the available data, researchers investigating potential chromatin associations can adapt established S. pombe ChIP protocols :

• Cross-linking optimization:

  • Test different formaldehyde concentrations (1-3%) and incubation times (10-30 min)

  • For proteins with weaker DNA interactions, consider dual cross-linking with additional agents like disuccinimidyl glutarate

• Chromatin preparation:

  • Lyse cells using glass bead disruption in buffer containing 100 mM HEPES-KOH pH 7.5, 1% Triton X-100, 0.1% Na-deoxycholate, 1 mM EDTA, 140 mM NaCl, protease inhibitors, and phosphatase inhibitors

  • Optimize sonication to achieve chromatin fragments of ~500-1000 bp

  • Monitor fragmentation efficiency by agarose gel electrophoresis

• Immunoprecipitation conditions:

  • Test various antibody concentrations (typically 2-10 μg per IP)

  • Compare different bead types (Protein A/G for rabbit polyclonal antibodies)

  • Include appropriate controls: IgG control, input sample, and ideally a SPBC1198.03c deletion strain

• Analysis strategies:

  • Design qPCR primers for genomic regions of interest based on localization data

  • Calculate enrichment relative to control regions (e.g., 18S ribosomal DNA)

  • For genome-wide studies, consider ChIP-seq to identify all binding sites

Given the nuclear localization of SPBC1198.03c, ChIP experiments could provide valuable insights into its potential roles in chromatin organization or gene regulation.

What are the optimal protocols for detecting SPBC1198.03c using immunofluorescence microscopy?

Based on the known localization patterns of SPBC1198.03c to specific cellular structures (cell tip and barrier septum), the following optimized immunofluorescence protocol can be developed:

• Cell preparation:

  • Culture S. pombe cells to mid-log phase (OD600 = 0.5-0.8)

  • Fix with 3.7% formaldehyde for 30 minutes at room temperature

  • Wash cells 3× with PEM buffer (100 mM PIPES pH 6.9, 1 mM EGTA, 1 mM MgSO4)

• Cell wall digestion optimization:

  • Create spheroplasts using zymolyase (1 mg/ml) in PEMS (PEM + 1.2 M sorbitol)

  • Carefully monitor digestion by microscopy to prevent over-digestion

  • Critical step: Optimization of digestion time (typically 30-60 min) is essential for antibody accessibility

• Permeabilization and blocking:

  • Permeabilize with PEMS + 1% Triton X-100 for 5 minutes

  • Block with PEMBAL (PEM + 1% BSA, 0.1% sodium azide, 100 mM lysine HCl) for 30 minutes

• Antibody incubation parameters:

  • Primary antibody: Test dilution series (1:100 to 1:1000) of SPBC1198.03c antibody in PEMBAL

  • Incubate overnight at 4°C in a humid chamber

  • Secondary antibody: Use fluorophore-conjugated anti-rabbit IgG (1:500 dilution)

• Imaging considerations:

  • Include DAPI staining for nuclear visualization

  • Use appropriate filters to avoid bleed-through between channels

  • Pay particular attention to cell tips and septum regions for SPBC1198.03c signals

• Validation controls:

  • Negative control: SPBC1198.03c deletion strain

  • Antibody specificity control: Primary antibody omission

  • Positive control: If available, a GFP-tagged SPBC1198.03c strain

This protocol should be optimized for specific laboratory conditions and microscopy equipment.

How does the expression and localization of SPBC1198.03c change during cell cycle and meiosis?

To investigate cell cycle-dependent and meiotic expression patterns of SPBC1198.03c, researchers can employ the following methodological approaches:

• Cell cycle analysis:

  • Synchronize cells using methods such as lactose gradient centrifugation, nitrogen starvation followed by release, or cdc25-22 temperature-sensitive mutants

  • Collect samples at defined time points across the cell cycle

  • Analyze SPBC1198.03c protein levels by Western blot and localization by immunofluorescence

• Meiotic expression profiling:

  • Utilize the pat1-114 temperature-sensitive system for synchronous meiosis induction

  • Prepare pat1-114/pat1-114 diploid cells as described in previous studies

  • Sample collection at defined meiotic stages (typically hourly for 8-12 hours)

  • RNA analysis using qRT-PCR or RNA protection assays with specific probes

  • Protein expression analysis by Western blot using the SPBC1198.03c antibody

• Data analysis framework:

  • Normalize expression to appropriate reference genes/proteins (e.g., act1+ for RNA)

  • Compare expression patterns with known cell cycle and meiotic markers

  • Correlate changes in expression with cytological markers of meiotic progression

• Live-cell imaging option:

  • Generate strains expressing fluorescently tagged SPBC1198.03c

  • Perform time-lapse imaging during normal growth or meiotic induction

  • Quantify changes in protein concentration at specific cellular locations

While specific data on SPBC1198.03c expression during meiosis is not provided in the available information, this methodological approach would provide comprehensive insights into its regulation and potential functions during these cellular processes.

What approaches can be used to identify interaction partners of SPBC1198.03c?

To identify and validate protein interaction partners of SPBC1198.03c, several complementary approaches can be employed:

• Co-immunoprecipitation optimization:

  • Cell lysis: Use buffer containing 50 mM Tris-HCl pH 7.5, 0.2% Triton X-100, 0.5 mM EDTA, 20% glycerol, 100 mM NaCl, and protease/phosphatase inhibitors

  • Antibody immobilization: Conjugate SPBC1198.03c antibody to appropriate beads or use pre-coupled anti-rabbit IgG beads

  • Incubation conditions: Mix lysate with antibody-beads by inversion for 2 hours at 4°C

  • Wash stringency: Optimize to reduce non-specific interactions while maintaining true partners

  • Elution methods: Compare SDS-based elution vs. peptide competition

• Proximity-based approaches:

  • BioID fusion: Generate SPBC1198.03c-BioID fusion protein to biotinylate proximal proteins

  • APEX2 tagging: Alternative proximity labeling with shorter labeling time

  • Split-BiFC: For validating specific interactions in vivo

• Mass spectrometry analysis pipeline:

  • Sample preparation: Process immunoprecipitates for LC-MS/MS analysis

  • Data analysis: Use appropriate search algorithms and databases

  • Filtering criteria: Compare against controls to identify specific interactors

  • Validation: Confirm key interactions by reverse co-IP or other methods

• Genetics-based validation:

  • Synthetic genetic array (SGA) analysis to identify functional relationships

  • Phenotypic analysis of double mutants

  • Suppressor/enhancer screens

These approaches provide complementary data that together build a comprehensive picture of SPBC1198.03c's protein interaction network and cellular functions.

What functional domains are present in SPBC1198.03c and how do they affect epitope selection?

Although specific domain information for SPBC1198.03c is limited in the available data, researchers can employ the following methodological strategy to identify domains and optimize epitope selection:

• Bioinformatic domain prediction:

  • Analyze the primary sequence using tools such as:

    • SMART (Simple Modular Architecture Research Tool)

    • Pfam for conserved domain identification

    • InterPro for integrated protein signature recognition

  • Predict secondary structure elements (α-helices, β-sheets)

  • Identify transmembrane regions, signal peptides, and localization signals

• Structural considerations for epitope selection:

  • Predict protein surface exposure using algorithms like Emini Surface Accessibility

  • Analyze hydrophilicity profiles using Kyte-Doolittle plots

  • Identify regions with high predicted antigenicity (Jameson-Wolf antigenic index)

  • Avoid highly conserved domains if specificity between related proteins is desired

• Experimental epitope mapping:

  • Generate overlapping peptide arrays spanning the SPBC1198.03c sequence

  • Test antibody binding to identify specific recognition regions

  • Validate findings using recombinant protein fragments

• Cross-reactivity assessment:

  • Test antibody against related proteins in S. pombe

  • Evaluate specificity using SPBC1198.03c deletion strains as negative controls

  • Consider cross-species reactivity if studying conserved mechanisms

Understanding domain organization informs not only epitope selection but also hypotheses about protein function. For example, the localization of SPBC1198.03c to cell tips and the septum suggests potential domains involved in targeting to these structures, which may influence antibody accessibility in different experimental contexts.

How can SPBC1198.03c antibody be used in quantitative proteomic studies?

SPBC1198.03c antibody can be incorporated into several proteomic workflows to investigate its function and regulation:

• Immunoprecipitation-mass spectrometry (IP-MS):

  • Immunoprecipitate SPBC1198.03c and associated proteins using optimized IP conditions

  • Process samples for LC-MS/MS analysis using established protocols

  • Implement appropriate controls (IgG control, SPBC1198.03c deletion strain)

  • Data analysis: Compare identified proteins against background using statistical methods

  • Validation: Confirm key interactions by complementary methods

• Chromatin proteomics approaches:

  • Given the nuclear localization of SPBC1198.03c, chromatin proteomics may be informative

  • Adapt protocols from quantitative proteomic analysis of chromatin-bound proteins in S. pombe

  • Fractionate cells to isolate chromatin-bound proteins

  • Compare chromatin proteome between wild-type and SPBC1198.03c deletion strains

• Post-translational modification mapping:

  • Immunoprecipitate SPBC1198.03c under various conditions

  • Analyze by MS/MS to identify phosphorylation, ubiquitination, or other modifications

  • Investigate condition-dependent changes in modification patterns

• Absolute quantification strategy:

  • Develop a targeted MS assay (PRM or SRM) for SPBC1198.03c

  • Use stable isotope-labeled standard peptides for absolute quantification

  • Measure protein abundance changes across conditions or mutant strains

• Data analysis framework:

  • Implement appropriate statistical methods for comparison between conditions

  • Network analysis to place SPBC1198.03c in functional protein clusters

  • Pathway enrichment analysis to identify biological processes associated with SPBC1198.03c

These proteomic approaches provide complementary information about SPBC1198.03c function, regulation, and position within cellular networks.

How can researchers generate and validate SPBC1198.03c mutant strains?

For researchers investigating SPBC1198.03c function through mutational analysis, the following methodological framework can be implemented:

• Deletion strain construction:

  • Utilize PCR-based gene targeting with selection markers (e.g., ura4+, kan)

  • Design primers with ~80 bp homology to regions flanking SPBC1198.03c

  • Transform S. pombe using lithium acetate method as described in previous protocols

  • Select transformants on appropriate selective media

  • Confirm deletion by PCR across deletion junctions and Southern blotting

• Point mutation and domain deletion strategy:

  • Implement CRISPR/Cas9 system adapted for S. pombe

  • Alternatively, use PCR-based site-directed mutagenesis

  • Design repair templates containing desired mutations

  • For domain deletions, ensure in-frame fusion of remaining regions

  • Verification by sequencing

• Conditional systems implementation:

  • For essential functions or conditional phenotypes:

    • Regulatable promoter replacement (nmt1+ promoter series with varying strengths)

    • Auxin-inducible degron system for protein depletion

    • Temperature-sensitive alleles

• Fluorescent tagging strategy:

  • C- or N-terminal tagging with GFP or other fluorescent proteins

  • Consider linker optimization to maintain protein function

  • Verify fusion protein functionality by complementation tests

• Phenotypic validation framework:

  • Growth assays under various conditions (temperature, nutrients, stress)

  • Cell morphology analysis

  • Cell cycle progression assessment

  • Specific assays based on localization at cell tips and septum

  • Chromosome segregation analysis given potential nuclear functions

This comprehensive approach provides multiple avenues to investigate SPBC1198.03c function through complementary mutational strategies.

How does SPBC1198.03c compare to homologous proteins in other model organisms?

To establish functional relationships between SPBC1198.03c and homologous proteins in other organisms, researchers can employ the following analytical framework:

• Sequence-based homology identification:

  • BLAST/PSI-BLAST searches against protein databases

  • Hidden Markov Model (HMM) approaches for sensitive detection of remote homologs

  • Multiple sequence alignment to identify conserved residues and domains

  • Phylogenetic analysis to determine evolutionary relationships

• Structural comparison methodology:

  • Predict or determine three-dimensional structures

  • Structural alignment to identify conserved folding patterns

  • Analysis of conserved surface patches that may indicate functional sites

  • Comparison of electrostatic surface potentials

• Functional conservation assessment:

  • Comparative analysis of knockout/mutant phenotypes across species

  • Complementation studies (can homologs functionally substitute for each other?)

  • Comparison of protein-protein interaction networks

  • Subcellular localization comparison

• Expression pattern analysis:

  • Compare tissue/cell type-specific expression in multicellular organisms

  • Analyze condition-dependent expression changes across species

  • Identify conserved regulatory elements in promoter regions

• Data integration strategy:

  • Synthesize findings from multiple approaches

  • Weight evidence based on reliability of methods

  • Develop testable hypotheses about conserved functions

This comparative approach leverages evolutionary conservation to gain insights into SPBC1198.03c function, particularly when direct experimental data is limited.

What can we learn from cross-species conservation of SPBC1198.03c epitopes?

Analysis of epitope conservation across species provides valuable insights for both antibody development and evolutionary biology:

• Conservation mapping methodology:

  • Perform multiple sequence alignment of SPBC1198.03c homologs

  • Map regions recognized by the antibody onto the alignment

  • Quantify conservation scores for epitope regions

  • Identify invariant residues within epitopes

• Cross-reactivity prediction framework:

  • Based on epitope conservation, predict potential cross-reactivity with homologs

  • Evaluate cross-species conservation of three-dimensional epitope structure

  • Assess impact of non-conserved residues on antibody binding

• Experimental cross-reactivity assessment:

  • Test antibody reactivity against recombinant homologs from other species

  • Perform Western blot and immunoprecipitation using lysates from related yeasts

  • Quantify binding affinity differences between homologs

• Functional epitope analysis:

  • Determine if conserved epitopes correspond to functional domains

  • Assess whether antibody binding affects protein function

  • Use antibody as a probe for evolutionarily conserved interaction surfaces

• Application to evolutionary studies:

  • Use conservation patterns to infer selective pressures

  • Identify regions under purifying selection (highly conserved)

  • Detect regions under diversifying selection (highly variable)

What strategies can resolve inconsistent Western blot results with SPBC1198.03c antibody?

When encountering variable or unexpected results with SPBC1198.03c antibody in Western blotting, implement this systematic troubleshooting approach:

• Sample preparation optimization:

  • Compare different lysis methods: TCA extraction vs. mechanical disruption

  • Test various lysis buffers with different detergents and salt concentrations

  • Include appropriate protease and phosphatase inhibitors

  • Standardize protein quantification method and loading amount

  • Evaluate both denaturing and native conditions if protein complexes are relevant

• Electrophoresis parameter optimization:

  • Test different gel percentages to optimize resolution

  • Compare reducing vs. non-reducing conditions

  • Adjust running conditions (voltage, time, temperature)

  • Evaluate the impact of different sample buffers and heating conditions

• Transfer and detection optimization:

  • Compare wet and semi-dry transfer methods

  • Optimize transfer time and current based on protein size

  • Test different membrane types (PVDF vs. nitrocellulose)

  • Evaluate blocking reagents (BSA vs. milk) for optimal signal-to-noise ratio

  • Titrate primary antibody concentration (typically 1:500 to 1:5000)

  • Optimize secondary antibody dilution and incubation conditions

• Controls and validation:

  • Include positive control (recombinant protein if available)

  • Use SPBC1198.03c deletion strain as negative control

  • Consider epitope-tagged version as additional control

  • Perform peptide competition assay to confirm specificity

• Data analysis recommendations:

  • Always include loading controls (e.g., α-tubulin)

  • Use quantitative analysis software for densitometry

  • Implement appropriate normalization method

  • Report variability across replicates

This systematic approach identifies variables affecting Western blot performance and leads to a robust, reproducible protocol for SPBC1198.03c detection.

How can researchers troubleshoot non-specific binding in immunoprecipitation experiments?

Non-specific binding in immunoprecipitation with SPBC1198.03c antibody can be addressed through this systematic optimization framework:

• Pre-clearing optimization:

  • Implement pre-clearing step with beads alone

  • Test different pre-clearing durations (1-3 hours)

  • Optimize bead type and amount for pre-clearing

• Buffer composition adjustments:

  • Systematically test increasing salt concentrations (100-500 mM)

  • Evaluate different detergent types and concentrations

  • Add competing proteins (BSA, gelatin) to reduce non-specific interactions

  • Consider adding agents like glycerol to stabilize specific interactions

• Bead selection and blocking:

  • Compare different matrices (Sepharose, magnetic, agarose)

  • Test pre-blocking of beads with BSA or non-fat milk

  • Evaluate directly conjugated antibody vs. protein A/G-mediated capture

• Washing protocol optimization:

  • Implement increasingly stringent wash conditions

  • Test graduated washing with buffers of increasing stringency

  • Optimize number of washes and wash volume

  • Compare different handling methods to minimize bead loss

• Experimental controls implementation:

  • Include no-antibody control

  • Use non-specific IgG as negative control

  • Include SPBC1198.03c deletion strain as specificity control

  • Consider denaturing IPs for direct interactors vs. native conditions for complexes

This comprehensive approach systematically identifies and addresses sources of non-specific binding to optimize immunoprecipitation specificity.

What technical considerations are important when using SPBC1198.03c antibody in ELISA?

To optimize ELISA performance with SPBC1198.03c antibody, researchers should consider this detailed methodological framework:

• Antigen preparation optimization:

  • For direct ELISA: Use purified recombinant SPBC1198.03c

  • For sandwich ELISA: Test different capture antibodies or recombinant protein

  • Optimize coating concentration (typically 1-10 μg/ml)

  • Compare coating buffers (carbonate buffer pH 9.6 vs. PBS)

  • Evaluate coating temperature and duration (4°C overnight vs. 37°C for 2 hours)

• Blocking conditions assessment:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Optimize blocking time and temperature

  • Evaluate washing buffer composition (PBS-T concentration)

  • Determine optimal washing protocol (number of washes, volume, duration)

• Antibody dilution optimization:

  • Perform antibody titration (serial dilutions from 1:100 to 1:100,000)

  • Test different diluents (with/without blocking protein)

  • Optimize incubation time and temperature

  • For sandwich ELISA: Test different detection antibody combinations

• Detection system considerations:

  • Compare HRP vs. AP enzyme conjugates

  • Evaluate different substrate options (TMB, ABTS, pNPP)

  • Optimize substrate incubation time

  • Select appropriate stopping method and timing

• Validation and quality control:

  • Include standard curve with recombinant protein

  • Implement positive and negative controls

  • Assess intra- and inter-assay variability

  • Determine detection limit and dynamic range

These systematic optimization steps ensure maximum sensitivity and specificity for quantitative detection of SPBC1198.03c in ELISA formats.

How can SPBC1198.03c antibody be used in single-cell analysis techniques?

SPBC1198.03c antibody can be adapted for emerging single-cell techniques using the following methodological approaches:

• Single-cell immunofluorescence optimization:

  • Implement microfluidic cell capture devices for consistent processing

  • Optimize fixation for single cells (concentration, duration, temperature)

  • Develop automated imaging protocols with consistent parameters

  • Implement machine learning-based image analysis for quantification

  • Correlate SPBC1198.03c localization with cell cycle markers

• Flow cytometry adaptation:

  • Optimize cell permeabilization for intracellular staining

  • Test fixation methods compatible with antibody epitope

  • Develop co-staining protocols with cell cycle markers

  • Implement controls for autofluorescence and non-specific binding

  • Consider fluorescence-activated cell sorting (FACS) to isolate specific subpopulations

• Single-cell mass cytometry (CyTOF) implementation:

  • Metal-conjugate SPBC1198.03c antibody using commercial kits

  • Develop multiplexed panel with other proteins of interest

  • Optimize staining concentrations for balanced signal

  • Implement clustering and dimensionality reduction for data analysis

• Single-cell Western blotting approach:

  • Adapt protocols for microfluidic single-cell western platforms

  • Optimize lysis conditions for single cells

  • Determine detection limits for SPBC1198.03c at single-cell level

  • Correlate protein levels with phenotypic features

• Proximity ligation assay (PLA) for protein interactions:

  • Combine SPBC1198.03c antibody with antibodies against potential interactors

  • Optimize probe concentrations and hybridization conditions

  • Implement appropriate controls for specificity

  • Quantify interaction signals at single-cell resolution

These advanced techniques provide insights into cell-to-cell variability in SPBC1198.03c expression, localization, and interactions that might be masked in population-based analyses.

What approaches can integrate SPBC1198.03c research with genome-wide studies?

To place SPBC1198.03c in broader genomic contexts, researchers can implement these integrative approaches:

• ChIP-seq methodology:

  • Optimize ChIP protocol for SPBC1198.03c as described in section 2.1

  • Prepare libraries for next-generation sequencing

  • Implement appropriate controls (input DNA, non-specific IgG)

  • Analysis pipeline:

    • Quality control and read alignment to reference genome

    • Peak calling using appropriate algorithms

    • Motif discovery for potential DNA binding sites

    • Integration with gene expression data

• Genetic interaction mapping:

  • Synthetic genetic array (SGA) with SPBC1198.03c deletion

  • Quantify genetic interactions using growth phenotypes

  • Classify interactions (synthetic lethal, suppressor, etc.)

  • Network analysis to identify functional clusters

• Transcriptome analysis integration:

  • Compare RNA-seq profiles between wild-type and SPBC1198.03c mutant strains

  • Identify differentially expressed genes and enriched pathways

  • Correlate expression changes with potential direct and indirect effects

  • Integration with ChIP-seq data to identify direct regulatory targets

• Protein interaction network analysis:

  • Combine IP-MS data with published protein interaction databases

  • Network visualization and community detection

  • Identification of SPBC1198.03c in protein complexes

  • Integration with genetic interaction data

• Multi-omics data integration:

  • Develop computational framework to integrate diverse datasets

  • Implement machine learning approaches for pattern recognition

  • Construct predictive models of SPBC1198.03c function

  • Validate key predictions experimentally

This integrative approach places SPBC1198.03c in the context of cellular networks and reveals its position in broader biological processes.