SPBC1921.04c Antibody

Basic Research Questions

• What is SPBC1921.04c and why is it significant in S. pombe research?

  SPBC1921.04c is a gene in Schizosaccharomyces pombe (fission yeast) that has been identified as one of the Mmi1 target RNAs. According to RNA-seq analysis, it's significantly affected by Rrp6 deletion, with an rrp6-cat/rrp6Δ ratio of 0.33 . This indicates it's regulated by the exosome complex through RNA degradation mechanisms involving the Rrp6 ribonucleolytic subunit. The protein is part of the broader network of mRNAs that may be targeted to the exosome via Rrp6 and RNA-binding proteins, making antibodies against this protein valuable for studying post-transcriptional regulatory mechanisms in yeast.

• What are the common applications for SPBC1921.04c antibody in basic research?

  SPBC1921.04c antibody is primarily used for:

  • Western blotting to detect protein expression levels

  • Immunoprecipitation to study protein-protein interactions

  • Immunofluorescence to visualize protein localization

  • ChIP assays to study protein-DNA interactions if the protein has DNA-binding properties

  These applications allow researchers to investigate the role of this protein in various cellular processes, particularly in RNA metabolism pathways involving the exosome complex and Mmi1-mediated RNA degradation.

• What controls should be included when using SPBC1921.04c antibody in experiments?

  Essential controls include:

  • Positive control: Wild-type S. pombe lysate known to express SPBC1921.04c

  • Negative control: Lysate from SPBC1921.04c deletion strain

  • Loading control: Antibody against a constitutively expressed protein (e.g., tubulin)

  • Pre-immune serum control: To assess background binding

  • Peptide competition assay: To verify specificity by pre-incubating the antibody with the immunizing peptide

  These controls help validate experimental results and ensure the antibody is specifically detecting the target protein.

Methodological Considerations

• What are the optimal conditions for Western blotting with SPBC1921.04c antibody?

  For optimal Western blotting results:

  • Sample preparation: Lyse cells in buffer containing protease inhibitors; heat samples at 95°C for 5 minutes in loading buffer

  • Gel percentage: 10-12% SDS-PAGE is appropriate for most S. pombe proteins

  • Transfer: Use PVDF membrane with standard transfer buffer

  • Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Primary antibody: Dilute SPBC1921.04c antibody 1:1000 in blocking buffer; incubate overnight at 4°C

  • Washing: 3 × 10 minutes with TBST

  • Secondary antibody: Anti-species HRP-conjugated antibody at 1:5000 for 1 hour at room temperature

  • Detection: Use ECL substrate with appropriate exposure time

  Optimize these conditions based on your specific experimental setup and antibody characteristics.

• How can I troubleshoot weak or no signal when using SPBC1921.04c antibody?

  If experiencing weak or no signal:

  1. Increase protein concentration in samples

  2. Optimize antibody concentration (try 1:500 dilution)

  3. Extend primary antibody incubation time to overnight at 4°C

  4. Use a more sensitive detection method (e.g., enhanced chemiluminescence)

  5. Check if protein is expressed under your experimental conditions

  6. Verify that the epitope is not masked by post-translational modifications

  7. Ensure protein transfer to membrane was successful using total protein stain

  8. Consider extracting protein under native conditions if epitope is conformation-dependent

  Systematic troubleshooting of each step will help identify the source of the problem.

Advanced Research Applications

• How can SPBC1921.04c antibody be used to study the relationship between exosome function and RNA processing in S. pombe?

  To investigate exosome-mediated RNA processing:

  1. Co-immunoprecipitation studies: Use SPBC1921.04c antibody to pull down the protein and analyze associated RNA components and exosome subunits (Dis3, Rrp6).

  2. RNA-immunoprecipitation (RIP): Cross-link protein-RNA complexes, immunoprecipitate with SPBC1921.04c antibody, and identify bound RNAs by RT-PCR or sequencing.

  3. Comparative analysis in exosome mutants: Compare SPBC1921.04c protein levels in wild-type, rrp6Δ, rrp6-cat, and dis3-4 mutant backgrounds (as shown in research examining the rrp6-cat/rrp6Δ ratio of 0.33) .

  4. Temporal analysis: Examine changes in SPBC1921.04c protein levels during cell cycle progression or in response to stress conditions to understand dynamic regulation by the exosome.

  These approaches can reveal whether SPBC1921.04c is directly involved in exosome function or is itself regulated by exosome-mediated RNA degradation pathways.

• What are the considerations for generating phospho-specific SPBC1921.04c antibodies to study its post-translational regulation?

  For phospho-specific antibody development:

  1. Phosphorylation site prediction: Use bioinformatics tools to predict potential phosphorylation sites and select those conserved across species or located in functional domains.

  2. Phosphopeptide design: Design phosphopeptides containing the predicted phosphorylation site with flanking amino acids (~10-15 aa total).

  3. Antibody generation: Immunize rabbits or other host animals with the phosphopeptide conjugated to a carrier protein.

  4. Validation strategies:

     • Test against phosphorylated and non-phosphorylated peptides

     • Compare reactivity with samples treated with/without phosphatase

     • Test specificity in kinase mutant strains

     • Verify with mass spectrometry data confirming the phosphorylation site

  5. Experimental applications: Use the phospho-specific antibody to study how the protein's phosphorylation state changes in response to cell cycle progression, stress conditions, or in kinase/phosphatase mutant backgrounds.

• How can SPBC1921.04c antibody be integrated into ChIP-seq or CUT&RUN protocols to study chromatin association?

  For chromatin immunoprecipitation applications:

  1. ChIP-seq optimization:

     • Crosslinking: Test both formaldehyde (1%, 10 min) and dual crosslinking (DSG followed by formaldehyde)

     • Sonication: Optimize to achieve 200-500 bp fragments

     • IP conditions: Test different antibody concentrations, incubation times, and washing stringencies

     • Controls: Include input, IgG control, and positive control (antibody against known chromatin-associated protein)

  2. CUT&RUN adaptation:

     • Cell permeabilization: Optimize digitonin concentration for S. pombe

     • Antibody binding: Incubate permeabilized cells with SPBC1921.04c antibody overnight

     • pA-MNase digestion: Titrate enzyme concentration and digestion time

     • Fragment release: Optimize salt concentration and incubation temperature

  3. Data analysis considerations:

     • Compare binding profiles to transcriptome data

     • Correlate with histone modification patterns

     • Analyze enrichment at Mmi1 target genes

     • Compare profiles in exosome mutant backgrounds

  These approaches can reveal if SPBC1921.04c has chromatin-associated functions, potentially linking RNA processing with transcriptional regulation.

Experimental Design Questions

• What is the appropriate experimental design to study SPBC1921.04c's role in the context of Mmi1-mediated RNA degradation?

  A comprehensive experimental approach should include:

  1. Genetic analysis:

     • Generate SPBC1921.04c deletion and conditional mutants

     • Create double mutants with mmi1, rrp6, and dis3 alleles

     • Analyze genetic interactions through growth assays at different temperatures

  2. Protein analysis:

     • Use SPBC1921.04c antibody to compare protein levels in wild-type vs. mmi1Δ, rrp6Δ strains

     • Perform co-immunoprecipitation to identify protein interactions with Mmi1 and exosome components

     • Analyze post-translational modifications by Western blot and mass spectrometry

  3. RNA analysis:

     • Measure SPBC1921.04c mRNA levels in mmi1Δ and exosome mutants by RT-qPCR

     • Perform transcriptome analysis (RNA-seq) in SPBC1921.04c mutants to identify affected pathways

     • Use RNA-immunoprecipitation to identify RNAs bound by SPBC1921.04c protein

  4. Microscopy:

     • Use SPBC1921.04c antibody for immunofluorescence to determine subcellular localization

     • Analyze co-localization with Mmi1 and exosome components

     • Track changes in localization during cell cycle or stress response

  This multifaceted approach will provide comprehensive insights into SPBC1921.04c's function in RNA metabolism pathways.

• How can I use the SPBC1921.04c antibody to investigate protein-protein interactions in different cellular compartments?

  To study compartment-specific interactions:

  1. Subcellular fractionation:

     • Separate nuclear, cytoplasmic, and membrane fractions

     • Perform Western blotting with SPBC1921.04c antibody on each fraction

     • Use compartment-specific markers (histone H3 for nucleus, tubulin for cytoplasm)

  2. Proximity labeling approaches:

     • Generate SPBC1921.04c fusion with BioID or TurboID

     • Perform biotin labeling followed by streptavidin pulldown

     • Validate interactions using SPBC1921.04c antibody

     • Identify interaction partners by mass spectrometry

  3. In situ approaches:

     • Perform proximity ligation assay (PLA) using SPBC1921.04c antibody and antibodies against potential interactors

     • Use fluorescence resonance energy transfer (FRET) with fluorophore-conjugated antibodies

     • Analyze co-localization by super-resolution microscopy

  4. Compartment-specific co-immunoprecipitation:

     • Perform IP with SPBC1921.04c antibody on fractionated samples

     • Compare interactome differences between compartments

     • Validate with reciprocal IPs using antibodies against identified partners

  These approaches will reveal how the protein's interactions differ across cellular compartments and how these might relate to compartment-specific functions.

Troubleshooting and Technical Considerations

• What are the optimal fixation and permeabilization conditions for immunofluorescence with SPBC1921.04c antibody in S. pombe?

  For optimal immunofluorescence:

  1. Cell wall digestion:

     • Treat cells with zymolyase (1 mg/ml) for 10-30 minutes at 37°C

     • Monitor digestion by checking for spheroplast formation

  2. Fixation options:

     • For general detection: 3.7% formaldehyde, 30 minutes at room temperature

     • For preserving nuclear structures: 3.7% formaldehyde + 0.2% glutaraldehyde

     • For membrane proteins: Methanol fixation (-20°C, 6 minutes)

  3. Permeabilization:

     • Standard: 0.1% Triton X-100 in PBS, 5 minutes

     • Gentle: 0.1% Saponin in PBS (reversible, good for membrane proteins)

     • Enhanced: 0.5% NP-40, 10 minutes (for nuclear proteins)

  4. Blocking and antibody incubation:

     • Block: 5% BSA or 5% normal serum from secondary antibody species

     • Primary antibody: 1:100 to 1:500 dilution, overnight at 4°C

     • Secondary antibody: 1:500 fluorophore-conjugated, 1 hour at room temperature

  5. Mounting and counterstaining:

     • DAPI (1 μg/ml) for nuclear counterstaining

     • Anti-fade mounting medium to prevent photobleaching

  Systematically test these conditions to determine optimal parameters for SPBC1921.04c detection.

• How can I validate the specificity of SPBC1921.04c antibody and address potential cross-reactivity issues?

  To validate antibody specificity:

  1. Genetic validation:

     • Compare wild-type vs. SPBC1921.04c deletion strains by Western blot

     • Use CRISPR/Cas9 knockout models if available

     • Test in strains with epitope-tagged SPBC1921.04c

  2. Biochemical validation:

     • Perform peptide competition assays

     • Test recognition of recombinant protein

     • Compare multiple antibodies targeting different epitopes

  3. Cross-reactivity assessment:

     • Perform BLAST analysis to identify proteins with similar epitopes

     • Test antibody against closely related species

     • Use mass spectrometry to identify all proteins in immunoprecipitates

  4. Application-specific validation:

     • For Western blot: Verify band size and pattern consistency

     • For IP: Confirm enrichment of target by Western blot or mass spectrometry

     • For IF: Verify localization pattern matches literature or epitope-tagged version

  Proper validation ensures reliable and reproducible results across different experimental applications.

Advanced Research Questions

• How can SPBC1921.04c antibody be used to investigate changes in protein dynamics during cellular stress responses?

  To study stress-induced changes:

  1. Stress induction protocols:

     • Oxidative stress: 0.5-1 mM H₂O₂, 15-60 minutes

     • Heat shock: 42°C, 10-30 minutes

     • Nutrient deprivation: Transfer to minimal media

     • DNA damage: 20-100 μg/ml phleomycin or 0.01% MMS

  2. Time-course analysis:

     • Collect samples at multiple timepoints (0, 15, 30, 60, 120 min)

     • Perform Western blotting with SPBC1921.04c antibody

     • Quantify changes in protein levels, normalized to loading control

  3. Post-translational modification analysis:

     • Use Phos-tag gels to detect phosphorylation changes

     • Perform IP followed by mass spectrometry to identify modifications

     • Use modification-specific antibodies if available

  4. Localization changes:

     • Perform immunofluorescence before and after stress

     • Track protein redistribution between compartments

     • Correlate with known stress response markers

  5. Protein stability assessment:

     • Cycloheximide chase experiments to measure protein half-life

     • Compare stability under normal vs. stress conditions

     • Analyze involvement of proteasome or autophagy pathways

  This comprehensive approach will reveal how SPBC1921.04c responds to cellular stress and may provide insights into its functional role during stress adaptation.

• What are the considerations for using SPBC1921.04c antibody in studying its potential role in the S. pombe cell cycle?

  For cell cycle studies:

  1. Synchronization methods:

     • Temperature-sensitive cdc25 block-release (G2/M)

     • Hydroxyurea arrest-release (S phase)

     • Lactose gradient centrifugation (size-based selection)

     • Nitrogen starvation/release (G1)

  2. Time-course experimental design:

     • Collect samples every 15-20 minutes for 1-2 cell cycles

     • Verify synchrony by flow cytometry or microscopy

     • Prepare protein extracts with phosphatase inhibitors to preserve modifications

  3. Analysis approaches:

     • Western blotting with SPBC1921.04c antibody to track protein levels

     • Co-immunoprecipitation to identify cell cycle-specific interactions

     • Immunofluorescence to track localization changes

     • Phospho-specific analysis using Phos-tag gels or phospho-specific antibodies

  4. Comparative analysis:

     • Compare with established cell cycle markers (Cdc2, Cdc13)

     • Analyze in cell cycle mutant backgrounds

     • Correlate protein changes with transcriptome data

  5. Functional experiments:

     • Overexpression analysis during specific cell cycle phases

     • Conditional depletion/inactivation with phenotypic analysis

     • Genetic interactions with established cell cycle regulators

  This approach will establish whether SPBC1921.04c has a cell cycle-regulated expression pattern or function, potentially revealing new insights into cell cycle control mechanisms.

• How can I incorporate SPBC1921.04c antibody into proteomic workflows to study protein complex dynamics?

  For advanced proteomic applications:

  1. Immunoprecipitation-mass spectrometry (IP-MS):

     • Use SPBC1921.04c antibody for native IP

     • Analyze by LC-MS/MS to identify interacting partners

     • Compare interactome across different conditions

     • Perform quantitative proteomics using SILAC or TMT labeling

     Method	Advantages	Limitations
     Native IP	Preserves physiological interactions	May miss weak interactions
     Crosslinked IP	Captures transient interactions	May introduce artifacts
     SILAC-IP	Quantitative comparison	Requires metabolic labeling
     TMT-IP	Multiplexing capability	Ratio compression issues

  2. Protein complex purification:

     • Sequential IP approach (tandem affinity purification)

     • Size exclusion chromatography followed by Western blotting

     • Blue native PAGE for intact complex analysis

     • Density gradient centrifugation with fraction analysis

  3. Structural proteomics:

     • Hydrogen-deuterium exchange MS to map interaction surfaces

     • Cross-linking MS to identify spatial proximity within complexes

     • Limited proteolysis to identify protected regions

     • Native MS for intact complex analysis

  4. Dynamic interactome analysis:

     • BioID or APEX proximity labeling with quantitative MS

     • Pulse-chase SILAC to measure interaction kinetics

     • Comparative analysis across cell cycle phases or stress conditions

     • Correlation with RNA-binding profiles (if applicable)

  These proteomic approaches will provide detailed insights into SPBC1921.04c protein complexes and their functional significance.