SPBC6B1.03c Antibody

What is SPBC6B1.03c and what are its known functions in fission yeast?

SPBC6B1.03c encodes an uncharacterized protein (UniProt ID: O43067) in Schizosaccharomyces pombe (fission yeast strain 972/ATCC 24843). The protein, also known as YGA3_SCHPO, is 272 amino acids in length and localizes to both the cytoplasm and nucleus . While its precise function remains to be fully elucidated, research indicates its potential involvement in chromatin organization and transcriptional regulation pathways.

Based on network analysis and genetic interaction studies, SPBC6B1.03c appears in datasets involving synthetic lethal interactions with human UBA1 (ubiquitin-like modifier activating enzyme 1), suggesting it may play a role in ubiquitin-mediated proteolysis or related cellular processes . This gene was identified in screens using the Yeast Augmented Network Analysis (YANA) approach, which examines genetic interactions to build functional networks.

What validation methods should I use to confirm SPBC6B1.03c antibody specificity?

When validating SPBC6B1.03c antibodies, implement a multi-technique approach:

• Western blot analysis with proper controls:

  • Use wild-type S. pombe lysates alongside a SPBC6B1.03c deletion strain

  • Include recombinant SPBC6B1.03c protein as a positive control

  • Test cross-reactivity with related proteins if known

• Immunoprecipitation followed by mass spectrometry:

  • Confirm the identity of immunoprecipitated proteins by peptide mapping

  • Compare results with datasets from unrelated antibodies to identify nonspecific binding

• Immunofluorescence correlation:

  • Compare antibody signal with GFP-tagged SPBC6B1.03c expression patterns

  • Verify cellular localization (both cytoplasmic and nuclear as expected)

• Genetic validation:

  • Test antibody reactivity in SPBC6B1.03c knockout or knockdown strains

  • Verify signal reduction or elimination in genetic deletion models

Similar validation approaches have been successfully used for other yeast proteins, as demonstrated in studies of S6K2 and CD36 antibodies.

What are the recommended applications for SPBC6B1.03c antibodies?

Based on successful applications of antibodies against similar yeast proteins, SPBC6B1.03c antibodies may be suitable for:

When optimizing these applications, reference protocols similar to those described for other nuclear/cytoplasmic proteins in yeast studies .

How can SPBC6B1.03c antibodies be integrated into studies of chromatin remodeling in fission yeast?

SPBC6B1.03c may be involved in chromatin organization based on genetic interaction data . To investigate its role in chromatin remodeling:

• Chromatin Immunoprecipitation (ChIP) analysis:

  • Perform ChIP-seq to identify genome-wide binding sites

  • Compare binding profiles with known chromatin remodelers like Hrp3

  • Analyze co-occupancy with histone modifications (H3K9Ac, H3K14Ac)

• Co-immunoprecipitation with chromatin remodeling complexes:

  • Use SPBC6B1.03c antibodies to pull down associated proteins

  • Analyze interactions with known chromatin remodelers (e.g., Hrp3, Mit1)

  • Validate interactions with reciprocal IPs

• Nucleosome positioning analysis:

  • Compare nucleosome occupancy in wild-type vs. SPBC6B1.03c mutant strains

  • Map SPBC6B1.03c binding relative to nucleosome-depleted regions

• Effect on antisense transcription:

  • Similar to studies with Hrp3 , analyze whether SPBC6B1.03c affects cryptic antisense transcription

  • Perform strand-specific RNA-seq in mutant vs. wild-type strains

Research on chromatin remodelers like Hrp3 has demonstrated that these proteins can organize nucleosome positioning within transcribed regions and suppress antisense transcription at euchromatic loci, potentially in concert with histone modifiers like Set2 and HDAC complexes .

What approaches can I use to analyze potential post-translational modifications of SPBC6B1.03c?

While current dbPTM data indicates no documented PTM records for YGA3_SCHPO (SPBC6B1.03c) , this likely reflects a gap in research rather than absence of modifications. To investigate potential PTMs:

• Immunoprecipitation followed by mass spectrometry:

  • Use SPBC6B1.03c antibodies to immunoprecipitate the native protein

  • Perform MS/MS analysis to identify modifications

  • Compare PTM profiles under different cellular conditions (e.g., nutrient stress, cell cycle phases)

• Phosphorylation-specific analysis:

  • Treat samples with phosphatase before Western blotting to identify mobility shifts

  • Use Phos-tag gels to separate phosphorylated isoforms

  • Develop phospho-specific antibodies if key sites are identified

• Ubiquitination analysis:

  • Perform IP under denaturing conditions to preserve ubiquitin modifications

  • Probe with anti-ubiquitin antibodies

  • Use ubiquitin remnant profiling (K-ε-GG) for site identification

• Site-directed mutagenesis validation:

  • Mutate putative modification sites and analyze effects on protein function

  • Compare localization and activity of wild-type vs. mutant proteins

What are optimal protocols for using SPBC6B1.03c antibodies in co-immunoprecipitation experiments?

For successful co-immunoprecipitation studies with SPBC6B1.03c antibodies:

Optimized Co-IP Protocol:

• Cell lysis and extract preparation:

  • Harvest mid-log phase S. pombe cells (OD₅₉₅ = 0.4-0.6)

  • Lyse cells using FastPrep bead beater in non-denaturing buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitors)

  • Clear lysate by centrifugation (13,000 × g, 15 min, 4°C)

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

• Immunoprecipitation:

  • Incubate 1 mg of pre-cleared lysate with 2-5 μg of SPBC6B1.03c antibody overnight at 4°C with gentle rotation

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

  • Wash beads 4× with lysis buffer and 2× with PBS

  • Elute bound proteins with SDS sample buffer or use native elution for downstream applications

• Analysis of interacting partners:

  • Separate eluted proteins by SDS-PAGE

  • Analyze by Western blot for suspected interacting partners

  • For unbiased discovery, perform mass spectrometry analysis

• Controls to include:

  • IgG control antibody IP

  • IP from SPBC6B1.03c deletion strain

  • Reciprocal IP of identified interacting partners

This approach is similar to methods used successfully for other nuclear and cytoplasmic yeast proteins .

How can SPBC6B1.03c antibodies be used to investigate genetic interaction networks?

SPBC6B1.03c has been identified in genetic screens using the YANA (Yeast Augmented Network Analysis) approach . To further explore its genetic interactions:

• Network validation by Co-IP:

  • Use SPBC6B1.03c antibodies to verify physical interactions with proteins encoded by synthetic lethal or enhancer genes

  • Validate protein-protein interactions predicted by genetic networks

• ChIP-seq correlation:

  • Compare SPBC6B1.03c binding sites with those of genetically interacting factors

  • Identify shared target genes or chromatin regions

• Pathway analysis with double mutants:

  • Create double mutants of SPBC6B1.03c with synthetic interactors

  • Use antibodies to assess protein levels and localization in these backgrounds

• Developing a physical interaction map:

  • Use affinity purification with SPBC6B1.03c antibodies followed by mass spectrometry (AP-MS)

  • Compare physical interaction network with genetic interaction data

  • Identify discrepancies between genetic and physical networks for further study

The YANA approach demonstrated with UBA1 provides a model for how antibodies can help bridge genetic and physical interaction maps:

What are the key considerations for developing SPBC6B1.03c-specific monoclonal antibodies?

When developing monoclonal antibodies against SPBC6B1.03c, consider:

• Antigen design strategy:

  • Select unique epitopes with low homology to other proteins

  • Consider using multiple peptide regions (N-terminal, middle, and C-terminal)

  • For full-length protein antigens, ensure proper folding through:

    • Expression in eukaryotic systems

    • Verification of structural integrity by circular dichroism

• Hybridoma screening methodology:

  • Implement a multi-tiered screening approach:

    • Initial ELISA against recombinant antigen

    • Secondary screening by Western blot with yeast extracts

    • Tertiary validation with immunoprecipitation

  • Include SPBC6B1.03c knockout controls in screening process

• Clone selection criteria:

  • Prioritize clones recognizing native protein in multiple applications

  • Test cross-reactivity with related proteins

  • Assess performance across different fixation conditions for microscopy applications

This approach mirrors successful monoclonal antibody development strategies used for other proteins, such as S6K2 and staphylococcal enterotoxin B (SEB) .

How should I troubleshoot non-specific binding when using SPBC6B1.03c antibodies in immunofluorescence?

When encountering non-specific binding in immunofluorescence applications:

• Optimize blocking conditions:

  • Test different blocking agents:

    • 5% BSA in PBS

    • 5-10% normal serum (species different from antibody host)

    • Commercial blocking solutions

  • Extend blocking time to 2 hours at room temperature

• Adjust antibody dilution and incubation parameters:

  • Perform titration series (1:100 to 1:2000)

  • Test overnight incubation at 4°C vs. 2 hours at room temperature

  • Include 0.1-0.3% Triton X-100 in antibody dilution buffer

• Modify fixation protocol:

  • Compare methanol fixation vs. 4% paraformaldehyde

  • Test gentle fixation (2% paraformaldehyde for 10 minutes)

  • For yeast cells, optimize cell wall digestion conditions

• Implement additional controls:

  • Pre-absorb antibody with recombinant antigen

  • Use SPBC6B1.03c deletion strains as negative control

  • Include peptide competition assays

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Test fluorophores with different excitation/emission spectra to avoid autofluorescence

These troubleshooting approaches have been successfully applied to other challenging antibodies in yeast studies and can be adapted for SPBC6B1.03c.

What are the recommended approaches for studying SPBC6B1.03c in heterochromatin regulation?

Based on findings that chromatin remodelers like Hrp3 influence heterochromatin silencing , investigate SPBC6B1.03c's potential role using:

• ChIP analysis at heterochromatic regions:

  • Design primers for centromeric repeats (dg/dh)

  • Compare SPBC6B1.03c binding in wild-type vs. RNAi-deficient strains

  • Analyze co-occupancy with heterochromatin proteins (e.g., Swi6/HP1)

• Analysis of heterochromatic silencing:

  • Use reporter strains with markers inserted in heterochromatic regions

  • Compare silencing in wild-type vs. SPBC6B1.03c mutant backgrounds

  • Assess SPBC6B1.03c antibody staining patterns at heterochromatic foci

• Interaction with RNAi machinery:

  • Perform co-IPs with RNAi components (Ago1, Dcr1)

  • Analyze whether SPBC6B1.03c associates with centromeric siRNAs

  • Test genetic interactions with RNAi pathway mutants

• Nucleosome stability assay:

  • Apply MNase digestion assay similar to that used for Hrp3

  • Compare chromatin accessibility in wild-type vs. mutant strains

  • Analyze SPBC6B1.03c binding relative to nucleosome positioning

Research on Hrp3 demonstrated its importance for stable chromatin structure and regulation of non-coding transcription in heterochromatic regions , providing a methodological framework for similar studies with SPBC6B1.03c.

Can SPBC6B1.03c antibodies be used to study orthologs in other yeast species?

When considering cross-species applications of SPBC6B1.03c antibodies:

• Sequence conservation analysis:

  • Perform sequence alignment of SPBC6B1.03c with potential orthologs

  • Focus on epitope regions recognized by the antibody

  • Calculate percent identity and similarity across species

• Cross-reactivity testing:

  • Prepare protein extracts from multiple yeast species

  • Perform Western blot analysis with SPBC6B1.03c antibody

  • Compare band patterns and intensities

• Epitope mapping considerations:

  • If the antibody is epitope-mapped, check conservation of specific epitopes

  • For polyclonal antibodies, consider affinity purification against conserved peptides

• Functional domain targeting:

  • Antibodies against highly conserved functional domains may show better cross-reactivity

  • Target antibodies to regions with >70% amino acid identity

Similar cross-species antibody applications have been demonstrated for CD36/SR-B3 and MARCH6 , where antibodies recognized proteins across multiple species despite some sequence divergence.

How can I adapt ChIP-seq protocols for SPBC6B1.03c studies?

For optimal ChIP-seq results with SPBC6B1.03c antibodies:

• Chromatin preparation:

  • Cross-link S. pombe cells with 1% formaldehyde for 15 minutes at room temperature

  • Quench with 125 mM glycine for 5 minutes

  • Lyse cells using glass bead disruption in lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitors)

  • Sonicate to generate fragments of 200-500 bp

• Immunoprecipitation optimization:

  • Use 5-10 μg of SPBC6B1.03c antibody per sample

  • Include IgG control and input samples

  • Perform overnight incubation at 4°C

  • Wash with increasing stringency buffers to reduce background

• Library preparation considerations:

  • Use carrier DNA/RNA for low-yield samples

  • Perform size selection (150-300 bp) before adapter ligation

  • Include spike-in controls for normalization

• Data analysis approach:

  • Normalize to input and IgG control

  • Use narrow peak calling for site-specific binding

  • Apply broader peak detection if functioning as a chromatin remodeler

  • Compare binding profiles with nucleosome positioning data

This approach builds on ChIP methods successfully used for chromatin factors in fission yeast studies .

What are the best experimental designs to study SPBC6B1.03c's potential role in cell stress responses?

To investigate SPBC6B1.03c's involvement in stress responses:

• Stress condition panels:

  • Expose cells to diverse stressors:

    • Oxidative stress (H₂O₂)

    • Nutrient limitation

    • Temperature shifts

    • DNA damage (UV, MMS)

    • Osmotic stress

  • Monitor SPBC6B1.03c protein levels and localization using the antibody

• Temporal analysis:

  • Perform time-course experiments following stress induction

  • Analyze changes in:

    • Protein abundance

    • Subcellular localization

    • Post-translational modifications

    • Protein-protein interactions

• Genetic interaction assessment:

  • Create double mutants with known stress response factors

  • Analyze epistatic relationships

  • Use antibodies to monitor protein expression in these backgrounds

• Chromatin association dynamics:

  • Perform ChIP under different stress conditions

  • Identify condition-specific binding sites

  • Compare with transcriptional changes using RNA-seq

This experimental design framework has been applied successfully to study stress responses in yeast and could be adapted for SPBC6B1.03c investigations.

How can SPBC6B1.03c antibodies be integrated with emerging single-cell technologies?

To leverage SPBC6B1.03c antibodies in single-cell analyses:

• Single-cell Western blotting:

  • Adapt protocols to work with individual yeast cells

  • Optimize lysis conditions to maintain epitope integrity

  • Calibrate detection sensitivity for low abundance proteins

• Mass cytometry (CyTOF) applications:

  • Conjugate SPBC6B1.03c antibodies with rare earth metals

  • Combine with antibodies against cell cycle markers

  • Analyze protein abundance across heterogeneous populations

• Spatial transcriptomics integration:

  • Combine antibody-based protein detection with RNA-FISH

  • Correlate SPBC6B1.03c protein localization with transcriptional states

  • Analyze spatial relationships with interacting partners

• Microfluidics-based approaches:

  • Design chips for single-cell isolation and analysis

  • Implement on-chip immunoassays for SPBC6B1.03c detection

  • Combine with live-cell imaging for temporal dynamics

These approaches build on emerging single-cell technologies that have been applied to study protein dynamics in other systems and could provide unprecedented insights into SPBC6B1.03c function.

What considerations should guide the development of SPBC6B1.03c nanobodies for advanced imaging applications?

Nanobodies offer advantages for intracellular tracking and super-resolution microscopy of SPBC6B1.03c:

• Design and production strategy:

  • Select unique epitopes with strong surface exposure

  • Express recombinant SPBC6B1.03c for immunization of camelids

  • Screen nanobody libraries using phage display

  • Test for minimal interference with protein function

• Validation approach:

  • Compare binding properties with conventional antibodies

  • Verify specificity using SPBC6B1.03c knockout controls

  • Assess performance in different fixation and permeabilization conditions

• Fluorophore conjugation:

  • Site-specific labeling at C-terminus

  • Test different fluorophore combinations for multicolor imaging

  • Optimize fluorophore-to-nanobody ratio

• Super-resolution applications:

  • Validate performance in STORM, PALM, or STED microscopy

  • Compare resolution with conventional antibodies

  • Develop exchange-PAINT compatible nanobodies for multiplexed imaging

These approaches build on successful nanobody development strategies used for cancer-related targets , which could be adapted for SPBC6B1.03c studies.

How can computational approaches enhance interpretation of SPBC6B1.03c antibody-based experimental data?

Integrate computational methods to maximize insights from SPBC6B1.03c antibody experiments:

• Network analysis of interactome data:

  • Apply graph theory to analyze protein-protein interaction networks

  • Identify network modules and hub proteins

  • Compare with genetic interaction networks from YANA studies

• Machine learning for antibody binding prediction:

  • Train algorithms to predict epitope accessibility in different conditions

  • Optimize antibody selection for specific applications

  • Predict cross-reactivity with related proteins

• Molecular dynamics simulations:

  • Model antibody-antigen interactions at atomic level

  • Predict effects of mutations on binding affinity

  • Optimize buffer conditions for specific applications

• Integration with multi-omics data:

  • Correlate antibody-based findings with:

    • Transcriptomic profiles

    • Chromatin accessibility data

    • Metabolomic changes

  • Build predictive models of SPBC6B1.03c function