SPBC543.05c Antibody

What is SPBC543.05c and what is its role in Schizosaccharomyces pombe?

SPBC543.05c is a protein coding gene in the fission yeast Schizosaccharomyces pombe (strain 972 / ATCC 24843), with UniProt accession number Q9HGM6. While its specific function has not been fully characterized in the literature, research in S. pombe has shown that many such proteins are involved in critical cellular processes including cell cycle regulation, microtubule organization, and phosphorylation-dependent signaling pathways.

Based on homology studies with other yeast proteins, SPBC543.05c may be involved in mitotic processes that regulate chromosome segregation, similar to other proteins that undergo cell cycle-dependent phosphorylation in S. pombe .

What applications has the SPBC543.05c antibody been validated for?

According to manufacturer specifications, the SPBC543.05c antibody (CSB-PA872576XA01SXV) has been validated for the following applications:

The antibody is raised in rabbit as a polyclonal antibody, using recombinant Schizosaccharomyces pombe (strain 972 / ATCC 24843) SPBC543.05c protein as the immunogen .

What is the recommended protocol for Western blotting with SPBC543.05c antibody?

For optimal Western blotting results with SPBC543.05c antibody:

• Extract S. pombe proteins using the TCA (trichloroacetic acid) method to ensure comprehensive protein extraction .

• Separate proteins by SDS-PAGE using 10% gels for optimal resolution of most S. pombe proteins.

• Transfer to nitrocellulose membranes.

• Block with 5% non-fat milk in PBST.

• Dilute primary SPBC543.05c antibody 1:1000 in blocking buffer and incubate overnight at 4°C.

• Wash membranes 3-5 times with PBST.

• Incubate with HRP-conjugated secondary antibody (anti-rabbit).

• Develop using an ECL chemiluminescence system.

For detecting phosphorylated forms of the protein, consider using Phos-tag SDS-PAGE, which has been successful for analyzing phosphorylation states of S. pombe proteins .

What are the optimal storage conditions for SPBC543.05c antibody?

The manufacturer recommends:

• Store upon receipt at -20°C or -80°C

• Avoid repeated freeze-thaw cycles

• The antibody is supplied in liquid form containing:

  • Preservative: 0.03% Proclin 300

  • Constituents: 50% Glycerol, 0.01M PBS, pH 7.4

Aliquoting the antibody into smaller volumes before freezing can help minimize freeze-thaw cycles and preserve activity .

How can I use SPBC543.05c antibody to study potential phosphorylation during the cell cycle?

To study cell cycle-dependent phosphorylation of SPBC543.05c:

• Synchronize cell population: Use one of these validated methods:

  • Temperature-sensitive cdc25-22 strain synchronization

  • Lactose gradient centrifugation for size-based separation

• Sampling strategy: Collect samples at specific cell cycle stages:

  • G1 phase (post-mitotic)

  • S phase

  • G2 phase

  • Mitosis (different stages)

• Detect phosphorylation changes:

  • Use Phos-tag SDS-PAGE to separate phosphorylated forms

  • Apply two-dimensional gel electrophoresis for complex phosphorylation patterns

  • Use phosphatase treatment as a control to confirm phosphorylation bands

• Quantification: For quantitative western blots, ensure:

  • Loading controls (α-tubulin is commonly used in S. pombe)

  • Standardized protein amount (50μg per lane recommended)

S. pombe proteins often show mobility shifts on SDS-PAGE when phosphorylated, as demonstrated with proteins like mal3p, where phosphorylation changes in a cell cycle-dependent manner with dephosphorylation at the interphase-mitosis transition and rephosphorylation at metaphase-anaphase transition .

What methods can I use to identify potential kinases that phosphorylate SPBC543.05c?

Based on studies of other S. pombe proteins, a systematic approach to identify potential kinases includes:

• In silico analysis:

  • Examine protein sequence for consensus phosphorylation motifs

  • Compare with known substrates of various kinases

• Kinase screening:

  • Test candidate kinases in vitro with recombinant protein

  • Use kinase-deficient strains to observe changes in phosphorylation state

• Phospho-specific antibody generation:

  • Generate antibodies against predicted phosphorylation sites

  • Validate with phosphomimetic mutants

• Genetic approaches:

  • Create a strain collection with various kinase mutants and examine SPBC543.05c phosphorylation status

  • Test for genetic interactions between SPBC543.05c and candidate kinases

Potential kinases to consider include:

• Cdc2 (CDK1 homolog, essential for cell cycle regulation)

• Sty1p (MAPK involved in stress responses)

• Plo1 (Polo-like kinase)

• Aurora kinase (Ark1p)

• Ssp2p (AMPK homolog)

How can I optimize SPBC543.05c antibody for immunofluorescence in S. pombe cells?

For successful immunofluorescence with SPBC543.05c antibody:

Combining microtubule visualization with SPBC543.05c localization may provide insights into its potential role in microtubule organization throughout the cell cycle .

What are the considerations for using SPBC543.05c antibody in chromatin immunoprecipitation (ChIP) experiments?

If investigating a potential role for SPBC543.05c in chromatin regulation:

• Cross-linking optimization:

  • Start with 1% formaldehyde for 15 minutes at room temperature

  • For transient interactions, try protein-protein crosslinkers like DSG before formaldehyde

• Chromatin fragmentation:

  • Sonication parameters: 15-20 cycles of 30 seconds ON/30 seconds OFF

  • Target fragment size: 200-500bp

  • Verify fragmentation efficiency by agarose gel

• Immunoprecipitation conditions:

  • Pre-clear chromatin with protein A/G beads

  • Incubate with 2-5μg SPBC543.05c antibody overnight at 4°C

  • Include IgG control and input samples

• Washing stringency:

  • Low salt, high salt, LiCl, and TE washes to reduce background

  • Monitor wash stringency to avoid signal loss

• Elution and reversal of cross-links:

  • SDS-containing buffer at 65°C

  • Proteinase K treatment

• Analysis options:

  • qPCR for known targets

  • ChIP-seq for genome-wide binding profile

The success of ChIP depends on epitope accessibility in the cross-linked chromatin, which varies depending on protein localization and interactions .

How can SPBC543.05c antibody be used to study protein-protein interactions in S. pombe?

For studying SPBC543.05c interactions:

• Co-immunoprecipitation (Co-IP):

  • Optimal lysis conditions: 50mM Tris-HCl pH 7.5, 150mM NaCl, 0.1% NP-40, with protease and phosphatase inhibitors

  • Cross-linking (optional): 1mM DSP for 30 minutes

  • Immunoprecipitate with 2-5μg SPBC543.05c antibody

  • Use mass spectrometry to identify interacting proteins

• Proximity labeling:

  • Create fusion protein of SPBC543.05c with BioID or TurboID

  • Validate function with complementation tests

  • Identify proteins in close proximity through streptavidin pulldown

• Yeast two-hybrid screening:

  • Create SPBC543.05c bait constructs

  • Screen against S. pombe cDNA library

  • Validate interactions with Co-IP

• Analytical techniques for complex detection:

  • Size exclusion chromatography followed by western blotting

  • Blue native PAGE for intact complex analysis

• Interaction visualization:

  • Proximity ligation assay (PLA) for in situ interaction detection

  • FRET microscopy with fluorescently tagged proteins

Based on studies of other S. pombe proteins, interactions with microtubule-associated proteins or cell cycle regulators would be particularly interesting to investigate .

How does SPBC543.05c expression and localization potentially change during mitosis to meiosis transition?

To investigate SPBC543.05c during mitosis-meiosis transition:

• Experimental setup:

  • Nitrogen starvation protocol to induce meiosis in h90 or h+/h- diploid cells

  • Time-course sampling before and after induction

  • Parallel protein and RNA extraction

• Expression analysis:

  • Western blotting with SPBC543.05c antibody

  • Quantitative RT-PCR for transcript levels

  • Normalize to known stable reference genes

• Localization studies:

  • Immunofluorescence at different stages

  • Live-cell imaging with fluorescently tagged SPBC543.05c

  • Co-localization with meiotic markers

• Functional studies:

  • Create SPBC543.05c deletion or conditional mutants

  • Analyze meiotic progression by DAPI staining

  • Assess spore formation and viability

During the mitosis-meiosis transition, many proteins undergo dramatic changes in expression, localization, and post-translational modifications. Since sexual differentiation in S. pombe occurs upon nutrient starvation, integrating stress response data with meiotic progression analysis may provide insights into SPBC543.05c regulation .

Why might I see multiple bands when using SPBC543.05c antibody in Western blots?

Multiple bands in Western blots can result from several factors:

• Post-translational modifications:

  • Phosphorylation (common in S. pombe cell cycle proteins)

  • Ubiquitination (potentially affecting protein levels during cell cycle)

  • Other modifications changing electrophoretic mobility

• Proteolytic degradation:

  • Add protease inhibitor cocktail to extraction buffer

  • Maintain samples at 4°C during processing

  • Consider adding N-ethylmaleimide to prevent deubiquitination

• Alternative splice variants or isoforms:

  • Verify against known transcript data for SPBC543.05c

  • Use RT-PCR to confirm presence of variants

• Cross-reactivity:

  • Test specificity using SPBC543.05c deletion strain

  • Consider epitope mapping to identify cross-reactive regions

For distinguishing phosphorylated forms specifically:

• Perform lambda phosphatase treatment on protein extracts

• Use Phos-tag SDS-PAGE for improved separation of phosphorylated species

• Consider 2D gel electrophoresis for complex modification patterns

How can I adapt flow cytometry protocols for studying SPBC543.05c in the S. pombe cell cycle?

Flow cytometry can be valuable for studying cell cycle-related proteins in S. pombe:

• Cell preparation challenges:

  • S. pombe cells form multimers, requiring careful gating strategies

  • Use light-scatter measurements to exclude cell doublets

  • G1 and G2 cells contain the same DNA content, complicating analysis

• Modified protocol for S. pombe cell cycle analysis:

  • Fix cells with 70% ethanol

  • For DNA content: stain with propidium iodide

  • For protein detection: use SPBC543.05c antibody with fluorescent secondary antibody

  • Measure both DNA content and width of DNA-associated fluorescence signal

• Gating strategy:

  • Discriminate between G1 and G2 phase by nuclear number (G1 cells contain two nuclei)

  • Use forward and side scatter to exclude cell clumps

  • Apply width vs. area gating for single-cell selection

• Data analysis approach:

  • Correlate SPBC543.05c signal intensity with cell cycle phase

  • Compare wild-type with mutant strains

  • Monitor changes through synchronized populations

This adapted approach addresses the unique challenges of S. pombe cell cycle analysis by flow cytometry and enables detection of cell cycle-regulated changes in SPBC543.05c .

What controls should I include when studying SPBC543.05c phosphorylation?

Robust controls for phosphorylation studies include:

• Negative controls:

  • SPBC543.05c deletion strain

  • Phosphatase-treated samples to remove all phosphorylation

  • Non-phosphorylatable mutants (S/T→A substitutions)

• Positive controls:

  • Treatment with phosphatase inhibitors (increases phosphorylation)

  • Phosphomimetic mutants (S/T→D/E substitutions)

  • Known phosphorylated protein as technical control

• Experimental validation controls:

  • Kinase inhibitor treatments

  • Temperature-sensitive kinase mutants

  • Synchronized vs. asynchronous cultures

• Technical controls for phospho-specific detection:

  • Phos-tag SDS-PAGE with and without Mn²⁺ ions

  • Anderson SDS-PAGE for superior resolution of modified proteins

  • Pre-absorption with phospho-peptides for antibody validation

For quantitative analysis:

• Include loading controls (α-tubulin recommended)

• Apply normalization between samples

• Consider using stable isotope labeling for mass spectrometry-based quantification

How can I use proteomics approaches with SPBC543.05c antibody to identify novel interactions?

Comprehensive proteomics strategies include:

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Native conditions: 50mM HEPES pH 7.5, 100mM KCl, 0.1% NP-40

  • Crosslinking: 1-2mM DSP for 30 minutes at room temperature

  • SPBC543.05c antibody coupled to magnetic beads

  • Analysis by LC-MS/MS (e.g., ThermoFisher LTQ)

• Proximity-dependent biotin identification (BioID):

  • Generate SPBC543.05c-BioID fusion expression construct

  • Validate functionality through complementation

  • Induce proximity labeling with biotin for 16-24 hours

  • Isolate biotinylated proteins with streptavidin

• Comparative analysis approach:

  • Compare interactomes under different conditions:

    • Different cell cycle stages

    • Nutrient stress vs. normal growth

    • Mitosis vs. meiosis

• Data analysis strategy:

  • Filter against common contaminants

  • Use emPAI values for protein abundance estimation

  • Apply stringent statistical thresholds for interaction confidence

  • Validate key interactions by reciprocal IP or other methods

These approaches have successfully identified protein complexes in S. pombe, such as in the characterization of mitochondrial proteins during G0 phase, showing the power of comprehensive proteomics in fission yeast research .

What are the considerations for generating and validating phospho-specific antibodies against SPBC543.05c?

For developing phospho-specific antibodies:

• Phosphosite identification strategy:

  • Analyze SPBC543.05c sequence for potential phosphorylation motifs

  • Perform phosphoproteomics to identify actual sites

  • Focus on sites conserved across species or in functional domains

• Peptide design parameters:

  • 10-15 amino acids surrounding the phosphorylation site

  • Include phosphorylated residue centrally

  • Consider coupling to carrier protein (KLH or BSA)

• Validation experiments:

  • Test against wild-type extracts vs. phosphatase-treated samples

  • Compare signals from non-phosphorylatable mutants

  • Perform peptide competition assays

  • Evaluate specificity with phosphomimetic mutants

• Application-specific validation:

  • Western blot: Test for consistent molecular weight band

  • Immunofluorescence: Verify expected localization pattern

  • Cell cycle analysis: Confirm cell cycle-dependent changes

• Benchmarking guidelines:

  • Signal should disappear with λ-phosphatase treatment

  • No signal should be detected in non-phosphorylatable mutants

  • Signal intensity should correlate with biological regulation

Based on studies of other S. pombe proteins like mal3p, where phosphorylation varies with cell cycle stages, phospho-specific antibodies can provide valuable insights into the regulation of SPBC543.05c .

How can I integrate SPBC543.05c studies with investigations of oxidative stress responses in S. pombe?

For investigating SPBC543.05c in oxidative stress responses:

• Experimental design:

  • Oxidant treatment options:

    • Hydrogen peroxide (0.07mM, 0.5mM, or 6mM)

    • Menadione (5mM)

    • tert-butylhydroperoxide (2mM)

  • Time course: 5-60 minutes post-treatment

  • Immediate cell harvesting by gentle centrifugation

• Response parameters to measure:

  • SPBC543.05c protein levels by Western blot

  • Phosphorylation state changes

  • Subcellular localization shifts

  • Transcriptional changes by qRT-PCR

• Stress pathway connections:

  • Test dependency on known stress regulators:

    • Sty1p-Atf1p MAPK pathway

    • Pap1p (AP-1-like factor)

    • Prr1p (two-component regulator)

• Phenotypic analysis:

  • Compare wild-type vs. SPBC543.05c mutant stress resistance

  • Spotting assays on oxidant-containing plates

  • Flow cytometry for cell cycle effects

  • Microscopy for morphological changes

This approach integrates methods from studies of oxidative stress responses in S. pombe, which have identified differential gene expression and protein modification patterns in response to various oxidants .

How might SPBC543.05c contribute to microtubule dynamics in S. pombe?

To investigate potential roles in microtubule regulation:

• Localization analysis:

  • Co-immunofluorescence with tubulin

  • Live imaging with GFP-tagged SPBC543.05c

  • Cell cycle stage-specific localization patterns

• Dynamic association measurements:

  • Fluorescence recovery after photobleaching (FRAP)

  • Single-molecule tracking

  • Correlate with microtubule growth/shrinkage phases

• Functional perturbation experiments:

  • Temperature-sensitive or deletion mutants

  • Overexpression studies

  • Phospho-mimetic and non-phosphorylatable variants

• Microtubule organization analysis:

  • Interphase microtubule arrays

  • Mitotic spindle formation and elongation

  • Post-anaphase array organization

• Drug sensitivity tests:

  • Methyl-2-benzimidazole-carbamate (MBC) resistance assays

  • Microtubule dynamics measurement in presence of drugs

For context, S. pombe microtubule-associated proteins like mal3p (EB1 homolog) show phosphorylation changes during cell cycle, particularly at transitions between interphase and mitosis, which affects microtubule stability and organization. Similar regulation might apply to SPBC543.05c if it has microtubule-related functions .

How can I integrate SPBC543.05c research with broader studies of the S. pombe phosphoproteome?

For comprehensive phosphoproteome integration:

• Experimental approaches:

  • SILAC labeling for quantitative phosphoproteomics

  • TiO₂ enrichment of phosphopeptides

  • Multiplexed kinase assays with recombinant proteins

• Comparative analysis framework:

  • Cell cycle phases (G1, S, G2, M)

  • Growth conditions (nutrient-rich vs. starvation)

  • Stress responses (oxidative, osmotic, heat shock)

  • Wild-type vs. kinase/phosphatase mutants

• Data integration strategies:

  • Map SPBC543.05c phosphosites to known kinase consensus motifs

  • Network analysis with known phosphorylation-dependent pathways

  • Cross-reference with interactome data

  • Integrate with transcriptome changes

• Functional categorization:

  • Signaling node identification

  • Temporal clustering of phosphorylation events

  • Correlation with physiological transitions

This approach has been successfully applied to understand how phosphorylation networks regulate critical processes in S. pombe, including the substantial reorganization during G0 entry and the oxidative stress response .

What bioinformatic resources are available to predict SPBC543.05c function and regulation?

Key bioinformatic resources include:

• Sequence analysis tools:

  • PomBase (https://www.pombase.org/) - S. pombe genome database

  • UniProt (Q9HGM6) - Protein information

  • PFAM - Domain prediction

  • PSIPRED - Secondary structure prediction

• Phosphorylation predictors:

  • NetPhos - General phosphorylation sites

  • GPS - Kinase-specific phosphorylation sites

  • DISPHOS - Disordered region phosphorylation

• Evolutionary analysis options:

  • OrthoDB - Orthology identification across species

  • MUSCLE/CLUSTAL - Multiple sequence alignment

  • FungiDB - Cross-species comparative genomics

• Network analysis resources:

  • STRING - Protein-protein interaction networks

  • KEGG - Pathway mapping

  • S. pombe specific interaction databases

• Expression data integration:

  • Gene Expression Omnibus (GEO)

  • ArrayExpress

  • PomBase expression viewer

These resources can help identify conserved regulatory elements, predict potential functions based on homology and domain architecture, and place SPBC543.05c within the broader context of S. pombe biology .

What are the emerging technologies for studying proteins like SPBC543.05c in single S. pombe cells?

Cutting-edge single-cell approaches include:

• Single-cell proteomics:

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

  • Microfluidic antibody capture for protein quantification

  • Single-cell Western blotting

• Advanced imaging methods:

  • Super-resolution microscopy (PALM/STORM) for protein localization

  • Single-molecule tracking of fluorescently tagged proteins

  • FRET/FLIM for protein-protein interactions in vivo

• Genetic manipulation at single-cell level:

  • CRISPR-Cas9 for precise genome editing

  • Optogenetic control of protein activity/localization

  • Degron systems for rapid protein depletion

• Single-cell multi-omics integration:

  • Paired transcriptome and proteome analysis

  • Correlation of genetic background with protein expression

  • Cell cycle position determination with protein dynamics

These approaches are particularly valuable for studying cell-to-cell variation in protein expression, localization, and modification, which may reveal heterogeneity in SPBC543.05c regulation not captured in population-level studies .