SPAC6G9.15c Antibody

What is SPAC6G9.15c and why is it relevant for S. pombe research?

SPAC6G9.15c is a gene locus in Schizosaccharomyces pombe (fission yeast) with the UniProt accession number Q92360 . As a component of the model organism S. pombe (strain 972/ATCC 24843), this gene and its protein product are studied to understand fundamental cellular processes. Antibodies against this protein enable researchers to investigate protein expression, localization, and function within the fission yeast model system, which has significant evolutionary conservation with higher eukaryotes including humans.

What validation methods should be employed before using SPAC6G9.15c antibody in experiments?

When validating SPAC6G9.15c antibody, researchers should implement a multi-step approach:

• Western blot analysis using wild-type S. pombe lysates alongside SPAC6G9.15c deletion mutants

• Immunoprecipitation followed by mass spectrometry to confirm target specificity

• Immunofluorescence microscopy comparing labeled patterns between wild-type and knockout strains

• Preabsorption tests with recombinant SPAC6G9.15c protein to demonstrate binding specificity

This methodological sequence resembles validation approaches used for other S. pombe antibodies, as demonstrated in studies of antibody specificity where researchers employ immunological techniques to confirm target binding before proceeding with experimental applications.

What are the optimal storage conditions for maintaining SPAC6G9.15c antibody activity?

For maximum stability and preserved immunoreactivity of SPAC6G9.15c antibody:

• Store antibody aliquots at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles (limit to <5 cycles)

• For working solutions, store at 4°C with antimicrobial preservatives (0.02% sodium azide)

• Monitor antibody activity periodically using positive control samples

• Follow manufacturer's recommendations for specific product formulations

These storage guidelines optimize antibody shelf-life and maintain consistent experimental performance, which is particularly important for specialized antibodies against low-abundance yeast proteins.

What are the recommended dilution protocols for SPAC6G9.15c antibody in different applications?

Note: Optimal dilutions should be determined empirically for each new lot of antibody and experimental setup. Start with the middle of the recommended range and adjust as needed based on signal intensity and background levels.

How can researchers optimize SPAC6G9.15c antibody performance in Western blot analysis?

To achieve robust and reproducible Western blot results with SPAC6G9.15c antibody:

• Sample preparation: Use fresh S. pombe cultures and efficient lysis methods (glass bead disruption in the presence of protease inhibitors)

• Protein denaturation: Heat samples at 95°C for 5 minutes in sample buffer containing SDS and reducing agents

• Gel electrophoresis: Utilize 10-12% polyacrylamide gels for optimal separation

• Transfer conditions: Employ semi-dry transfer at 15V for 30 minutes or wet transfer at 30V overnight at 4°C

• Blocking: Use 5% non-fat milk in TBS-T for 1 hour at room temperature

• Primary antibody incubation: Apply SPAC6G9.15c antibody at optimized dilution (1:1000 recommended starting point) in blocking buffer overnight at 4°C

• Washing: Perform 4-5 washes with TBS-T, 5 minutes each

• Secondary antibody: Use HRP-conjugated anti-species antibody at 1:5000 dilution for 1 hour at room temperature

• Detection: Apply enhanced chemiluminescence substrate and image using appropriate detection system

This methodological approach maximizes specificity while minimizing background, critical for detecting potentially low-abundance yeast proteins.

What protocol is recommended for immunofluorescence microscopy using SPAC6G9.15c antibody?

For optimal subcellular localization of SPAC6G9.15c protein in fission yeast:

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

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

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

• Digest cell walls with Zymolyase-100T (1 mg/ml) for 30 minutes at 37°C

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

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

• Incubate with SPAC6G9.15c antibody (1:200 dilution) in PEMBAL overnight at 4°C

• Wash 3× with PEMBAL

• Apply fluorophore-conjugated secondary antibody (1:500) for 2 hours at room temperature

• Wash 3× with PEMBAL

• Mount with antifade mounting medium containing DAPI

• Image using confocal microscopy with appropriate filter sets

This protocol has been adapted from established methods for immunolocalization in fission yeast, ensuring minimal autofluorescence and maximum signal-to-noise ratio.

How can SPAC6G9.15c antibody be effectively used in chromatin immunoprecipitation (ChIP) experiments?

For investigating potential DNA-protein interactions involving SPAC6G9.15c:

• Crosslink S. pombe cells with 1% formaldehyde for 15 minutes at room temperature

• Quench with 125 mM glycine for 5 minutes

• Lyse cells using mechanical disruption with glass beads

• Sonicate chromatin to generate fragments of 200-500 bp

• Pre-clear lysate with protein A/G beads for 1 hour at 4°C

• Incubate cleared lysate with SPAC6G9.15c antibody (5-10 μg per reaction) overnight at 4°C

• Add protein A/G beads and incubate for 2 hours at 4°C

• Wash sequentially with low-salt, high-salt, LiCl, and TE buffers

• Elute protein-DNA complexes and reverse crosslinks at 65°C overnight

• Treat with RNase A and Proteinase K

• Purify DNA using phenol-chloroform extraction or commercial kits

• Analyze by qPCR or next-generation sequencing

This protocol enables researchers to identify genomic regions associated with SPAC6G9.15c protein, providing insights into potential regulatory functions.

What strategies can researchers employ to study post-translational modifications of SPAC6G9.15c protein?

To investigate potential post-translational modifications (PTMs):

• Immunoprecipitate SPAC6G9.15c protein using the specific antibody

• Separate proteins by SDS-PAGE and detect with:

  • Phosphorylation-specific antibodies (anti-phosphoserine, anti-phosphothreonine, anti-phosphotyrosine)

  • Acetylation-specific antibodies (anti-acetyl-lysine)

  • Ubiquitination-specific antibodies (anti-ubiquitin)

• For comprehensive PTM mapping:

  • Perform immunoprecipitation using SPAC6G9.15c antibody

  • Subject purified protein to tryptic digestion

  • Analyze peptides by LC-MS/MS with data-dependent acquisition

  • Process data using PTM-focused search algorithms (e.g., MaxQuant, Proteome Discoverer)

• Validate identified PTMs through site-directed mutagenesis and functional assays

This comprehensive approach allows researchers to characterize the "modificome" of SPAC6G9.15c protein and understand its regulatory mechanisms.

How does cell cycle progression affect SPAC6G9.15c protein expression and localization?

To investigate cell cycle-dependent dynamics:

• Synchronize S. pombe cultures using:

  • Temperature-sensitive cdc mutants

  • Nitrogen starvation-release

  • Lactose gradient centrifugation

• Collect samples at defined time points spanning the cell cycle

• Process parallel samples for:

  • Western blot analysis with SPAC6G9.15c antibody to assess protein levels

  • Immunofluorescence microscopy to determine subcellular localization

  • RT-qPCR to monitor mRNA expression

• Correlate findings with cell cycle phase markers:

  • DNA content (flow cytometry)

  • Septation index (calcofluor staining)

  • Expression of known cell cycle-regulated genes

This multi-faceted approach provides insights into the temporal regulation and functional significance of SPAC6G9.15c throughout the cell division cycle.

What are common issues encountered with SPAC6G9.15c antibody and how can they be resolved?

These troubleshooting strategies address the most common technical challenges researchers face when working with specialized antibodies for yeast proteins.

What controls should be included in experiments using SPAC6G9.15c antibody?

To ensure experimental validity and interpretability:

• Positive controls:

  • Wild-type S. pombe lysates with known SPAC6G9.15c expression

  • Recombinant SPAC6G9.15c protein (if available)

  • S. pombe strains with tagged SPAC6G9.15c (e.g., GFP-fusion)

• Negative controls:

  • SPAC6G9.15c deletion mutant lysates

  • Primary antibody omission

  • Isotype control antibody (same species and isotype)

  • Competitive blocking with immunizing peptide

• Technical controls:

  • Loading control for Western blots (e.g., anti-tubulin)

  • Subcellular marker antibodies for colocalization studies

  • Serial dilution of lysates to establish linearity of detection

These controls enable researchers to distinguish specific signals from non-specific background and validate experimental findings.

How can researchers determine the interactome of SPAC6G9.15c protein using antibody-based approaches?

To identify protein-protein interactions involving SPAC6G9.15c:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate SPAC6G9.15c using the specific antibody

  • Analyze co-precipitated proteins by mass spectrometry

  • Validate interactions by reciprocal Co-IP and Western blotting

• Proximity-dependent biotin identification (BioID):

  • Generate BioID2-SPAC6G9.15c fusion constructs

  • Express in S. pombe and supply biotin

  • Affinity-purify biotinylated proteins and identify by mass spectrometry

  • Confirm proximity with SPAC6G9.15c antibody in conventional assays

• Fluorescence microscopy co-localization:

  • Perform dual immunofluorescence with SPAC6G9.15c antibody and antibodies against candidate interacting proteins

  • Analyze co-localization using confocal microscopy and appropriate statistical methods

• Cross-linking immunoprecipitation (CLIP):

  • Cross-link protein complexes in vivo

  • Immunoprecipitate with SPAC6G9.15c antibody

  • Identify cross-linked proteins by mass spectrometry

These complementary approaches provide a comprehensive view of the SPAC6G9.15c protein interaction network.

What methodologies can be employed to study SPAC6G9.15c protein dynamics during cellular stress responses?

To investigate stress-induced changes:

• Stress induction experimental design:

  • Expose S. pombe cultures to various stressors (oxidative stress, heat shock, nutrient limitation)

  • Collect samples at multiple time points (0, 15, 30, 60, 120 minutes)

  • Process for protein, RNA, and microscopy analyses

• Quantitative protein analysis:

  • Western blot with SPAC6G9.15c antibody for total protein levels

  • Subcellular fractionation to track protein redistribution

  • Phospho-specific detection to monitor stress-induced modifications

• Localization dynamics:

  • Live-cell imaging with fluorescent-tagged SPAC6G9.15c

  • Fixed-cell immunofluorescence with SPAC6G9.15c antibody

  • Quantitative image analysis to measure spatial redistribution

• Functional interrogation:

  • ChIP-seq before and after stress to identify stress-dependent DNA binding

  • RNA-seq in wild-type vs. SPAC6G9.15c mutants under stress conditions

  • Protein stability assays using cycloheximide chase

This systematic approach reveals how SPAC6G9.15c responds to cellular stress and contributes to stress adaptation mechanisms.

How can cross-species conservation of SPAC6G9.15c be investigated using antibody-based methods?

To explore evolutionary conservation:

• Cross-reactivity testing:

  • Test SPAC6G9.15c antibody against lysates from related yeast species (S. cerevisiae, C. albicans) and higher eukaryotes

  • Perform Western blot analysis with standardized protein loading

  • Evaluate binding to recombinant homologs from different species

• Structural conservation analysis:

  • Immunoprecipitate homologous proteins from different species

  • Subject to structural proteomic analysis (limited proteolysis, hydrogen-deuterium exchange)

  • Compare conformational epitopes using epitope mapping approaches

• Functional complementation:

  • Express SPAC6G9.15c homologs from different species in S. pombe deletion mutants

  • Assess rescue of phenotypes

  • Use SPAC6G9.15c antibody to confirm expression and proper localization

• Phylogenetic immunology:

  • Generate antibodies against conserved epitopes of SPAC6G9.15c

  • Test reactivity across species

  • Correlate antibody binding with functional conservation

These approaches provide insights into the evolutionary conservation of SPAC6G9.15c structure and function across species.

How can SPAC6G9.15c antibody be utilized in single-cell analysis techniques?

For investigating cell-to-cell variability:

• Single-cell Western blotting:

  • Separate individual S. pombe cells on microfluidic devices

  • Perform in situ cell lysis, protein separation, and immunoblotting

  • Detect SPAC6G9.15c protein using the specific antibody

  • Quantify expression levels in individual cells

• Mass cytometry (CyTOF):

  • Label SPAC6G9.15c antibody with rare earth metals

  • Stain fixed and permeabilized S. pombe cells

  • Analyze single-cell protein expression by time-of-flight mass spectrometry

  • Correlate with other cellular parameters

• Imaging flow cytometry:

  • Perform immunofluorescence with SPAC6G9.15c antibody

  • Analyze thousands of individual cells

  • Quantify protein levels and subcellular localization simultaneously

This integration of antibody-based detection with single-cell technologies reveals heterogeneity in SPAC6G9.15c expression and localization within genetically identical populations.

What approaches can researchers use to study the functional domains of SPAC6G9.15c protein?

To map functional regions:

• Domain-specific antibody generation:

  • Design peptide antigens corresponding to predicted functional domains

  • Generate and validate domain-specific antibodies

  • Compare binding patterns with the full-length SPAC6G9.15c antibody

• Deletion mutant analysis:

  • Create S. pombe strains expressing SPAC6G9.15c with specific domain deletions

  • Perform immunoprecipitation and Western blot with SPAC6G9.15c antibody

  • Correlate structural alterations with functional changes

• Protein interaction mapping:

  • Express individual domains as fusion proteins

  • Perform pull-down assays with potential interaction partners

  • Validate interactions using SPAC6G9.15c antibody in full-length context

• Conformational epitope mapping:

  • Use hydrogen-deuterium exchange mass spectrometry to identify antibody binding regions

  • Correlate epitope accessibility with protein conformational states

  • Map functional implications of conformational changes

These approaches provide insights into structure-function relationships of SPAC6G9.15c protein and guide targeted therapeutic interventions.