SPBC3B9.04 Antibody

What is SPBC3B9.04 and what cellular functions does it serve?

SPBC3B9.04 is classified as a predicted mitochondrial methyltransferase in Schizosaccharomyces pombe (fission yeast). The protein has a molecular weight of approximately 28,743 Da . As a methyltransferase, it likely plays a role in methylation reactions within the mitochondria, potentially affecting mitochondrial protein function, RNA processing, or metabolic regulation. Current research indicates that it is primarily localized to the mitochondria, though its precise functional characterization remains an active area of investigation.

What types of SPBC3B9.04 antibodies are commercially available?

Current commercial offerings include rabbit polyclonal antibodies against SPBC3B9.04. These antibodies are typically produced using a recombinant Schizosaccharomyces pombe (strain 972 / ATCC 24843) SPBC3B9.04 protein as the immunogen . They are provided as liquid formulations, often containing preservatives like 0.03% Proclin 300 and stabilizers such as 50% glycerol in PBS (pH 7.4) . These antibodies are specifically intended for research applications, not for diagnostic procedures.

What applications are SPBC3B9.04 antibodies validated for?

SPBC3B9.04 antibodies have been tested and validated for:

• Western blotting (WB) for protein detection and quantification

• Enzyme-linked immunosorbent assays (ELISA)

Researchers should verify specific application conditions when using these antibodies for other techniques such as immunoprecipitation, immunohistochemistry, or chromatin immunoprecipitation, as these may require additional validation.

How does the predicted function of SPBC3B9.04 relate to other mitochondrial methyltransferases in model organisms?

As a predicted mitochondrial methyltransferase in S. pombe, SPBC3B9.04 may have functional homologs in other model organisms like S. cerevisiae or higher eukaryotes. Mitochondrial methyltransferases can participate in various processes including protein modification, RNA methylation, and small molecule metabolism. Understanding SPBC3B9.04's evolutionary conservation may provide insights into fundamental mitochondrial functions across species. Comparative analyses using bioinformatics tools alongside experimental approaches with the SPBC3B9.04 antibody could help establish functional relationships with better-characterized methyltransferases in other organisms .

How can SPBC3B9.04 antibodies be used to investigate mitochondrial protein import and processing?

The study of mitochondrial protein import often requires specialized techniques to track protein localization and processing. Using SPBC3B9.04 antibodies in combination with subcellular fractionation protocols adapted for fission yeast could help investigate the import pathways for this methyltransferase. Based on protocols described for S. pombe mitochondrial isolation, researchers can perform in vitro import assays similar to those used for pre-Su9DHFR . Detection with SPBC3B9.04 antibodies via western blotting could reveal processing intermediates and mature forms, providing insights into the protein's import mechanism and potential post-translational modifications.

What is known about the regulation of SPBC3B9.04 during different cellular conditions, and how can antibodies help investigate this?

Understanding the regulation of SPBC3B9.04 under different cellular conditions (e.g., nutrient limitation, oxidative stress, cell cycle phases) would require quantitative analysis using validated antibodies. Techniques such as quantitative western blotting, combined with transcriptional analysis, could reveal whether regulation occurs primarily at the transcriptional or post-transcriptional level. Similar approaches have been used to investigate translational control in fission yeast, where genome-wide translational profiles were integrated with other data such as mRNA steady-state levels and RNA polymerase II occupancy . Applying these methodologies to SPBC3B9.04 could provide insights into its regulatory mechanisms.

What are the optimal conditions for using SPBC3B9.04 antibodies in western blotting?

For optimal western blotting results with SPBC3B9.04 antibodies:

• Sample preparation:

  • Use a reliable protein extraction method for fission yeast, such as mechanical disruption with glass beads in an appropriate lysis buffer

  • Include protease inhibitors to prevent degradation

  • For mitochondrial proteins, consider specific mitochondrial isolation protocols

• SDS-PAGE conditions:

  • Use 10-12% polyacrylamide gels for optimal resolution of the ~29 kDa protein

  • Include molecular weight markers spanning 15-50 kDa

• Transfer and blocking:

  • PVDF membranes are recommended for optimal protein binding

  • Block in 5% non-fat dry milk or BSA in TBST

• Antibody incubation:

  • Primary antibody dilution: Start with 1:1000 and optimize as needed

  • Incubate overnight at 4°C for best results

  • Secondary antibody: Anti-rabbit IgG-HRP at 1:5000 dilution

• Detection:

  • Enhanced chemiluminescence (ECL) is suitable for detection

  • For quantitative analysis, consider fluorescence-based secondary antibodies

Always include appropriate positive and negative controls to validate the specificity of the antibody binding .

How can the subcellular localization of SPBC3B9.04 be verified using immunofluorescence microscopy?

To verify the mitochondrial localization of SPBC3B9.04:

• Cell fixation and permeabilization:

  • Fix S. pombe cells with 3.7% formaldehyde for 30 minutes

  • Permeabilize cell walls with zymolyase (1 mg/ml) for 30 minutes at 37°C

  • Further permeabilize with 1% Triton X-100 for 2 minutes

• Antibody staining:

  • Block with 1% BSA in PBS for 1 hour

  • Incubate with SPBC3B9.04 primary antibody (1:100 to 1:500 dilution)

  • Use appropriate fluorescently-labeled secondary antibody

• Mitochondrial co-localization:

  • Co-stain with established mitochondrial markers (e.g., MitoTracker)

  • Alternatively, co-stain with antibodies against known mitochondrial proteins

• Imaging considerations:

  • Use high-resolution confocal microscopy for optimal results

  • Consider deconvolution to improve signal-to-noise ratio

  • Perform Z-stack imaging to capture the full volume of cells

• Controls:

  • Include a peptide competition assay to confirm antibody specificity

  • Use SPBC3B9.04 deletion strains as negative controls if available

This approach has been successfully applied for visualizing various fission yeast proteins, such as the spindle pole body component Cut12 .

What strategies can be employed to validate the specificity of SPBC3B9.04 antibodies?

Ensuring antibody specificity is critical for reliable results. Several validation strategies include:

• Genetic validation:

  • Test antibody reactivity in wild-type versus SPBC3B9.04 deletion strains

  • Use CRISPR/Cas9 or traditional homologous recombination to create knockout strains

• Overexpression testing:

  • Create strains overexpressing SPBC3B9.04 and confirm increased signal

  • Use inducible promoters (e.g., nmt1) to control expression levels

• Epitope tagging:

  • Create strains expressing tagged versions (e.g., HA, GFP) of SPBC3B9.04

  • Compare localization/detection patterns between tagged proteins and antibody staining

• Mass spectrometry validation:

  • Perform immunoprecipitation followed by mass spectrometry

  • Confirm that peptides corresponding to SPBC3B9.04 are detected

• Cross-reactivity assessment:

  • Test antibody against closely related proteins or paralogs

  • Perform peptide competition assays with immunizing peptide

These validation approaches should be documented when reporting experimental results using SPBC3B9.04 antibodies to ensure scientific rigor .

What are common issues when using SPBC3B9.04 antibodies and how can they be resolved?

Always prepare fresh samples and store the antibody according to manufacturer recommendations (typically -20°C or -80°C, avoiding repeated freeze-thaw cycles) .

How can SPBC3B9.04 antibody sensitivity be enhanced for detecting low abundance proteins?

For detecting low-abundance SPBC3B9.04 protein:

• Sample enrichment techniques:

  • Perform subcellular fractionation to isolate mitochondria

  • Use immunoprecipitation to concentrate the target protein

  • Consider using detergents optimized for mitochondrial membrane proteins

• Signal amplification methods:

  • Employ tyramide signal amplification (TSA) for immunofluorescence

  • Use high-sensitivity chemiluminescent substrates for western blots

  • Consider biotin-streptavidin amplification systems

• Detection optimization:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize antibody concentration through careful titration

  • Use signal enhancers compatible with your detection system

• Instrument settings:

  • For western blots, use longer exposure times or more sensitive imaging systems

  • For microscopy, adjust gain settings and exposure times appropriately

  • Consider super-resolution microscopy techniques for improved detection

Similar sensitivity enhancement approaches have been successfully used for detecting low-abundance proteins in fission yeast, such as components of the nuclear pore complex .

How do different fixation methods affect SPBC3B9.04 antibody performance in immunofluorescence?

The choice of fixation method can significantly impact antibody performance for mitochondrial proteins like SPBC3B9.04:

For optimal results with SPBC3B9.04 antibody, perform comparative studies with different fixation methods in your specific experimental system. Successful immunofluorescence studies of fission yeast proteins often use a combination of aldehyde fixation followed by careful enzymatic digestion of the cell wall .

How can SPBC3B9.04 antibodies be used to study protein-protein interactions involving this methyltransferase?

To investigate protein-protein interactions involving SPBC3B9.04:

• Co-immunoprecipitation (Co-IP):

  • Use SPBC3B9.04 antibody coupled to protein A/G beads

  • Extract proteins under native conditions to preserve interactions

  • Identify interacting partners by western blotting or mass spectrometry

  • Consider crosslinking approaches to capture transient interactions

• Proximity-based labeling:

  • Generate SPBC3B9.04 fusion with BioID or APEX2

  • Express in S. pombe and activate labeling

  • Purify biotinylated proteins and identify by mass spectrometry

  • Confirm interactions with SPBC3B9.04 antibody

• Fluorescence microscopy approaches:

  • Perform dual immunofluorescence with SPBC3B9.04 antibody and antibodies against candidate interacting proteins

  • Calculate co-localization coefficients to quantify spatial relationships

  • Consider FRET-based approaches if suitable fluorophore-conjugated antibodies are available

• Genetic interaction studies:

  • Combine with deletion/mutation of potential interacting partners

  • Use SPBC3B9.04 antibody to assess protein levels or localization changes

  • Look for synthetic phenotypes suggesting functional relationships

Similar approaches have been used to characterize protein complexes in fission yeast, such as the DASH complex and kinetochore components .

What insights can SPBC3B9.04 antibodies provide about mitochondrial function in fission yeast?

SPBC3B9.04 antibodies can provide valuable insights into mitochondrial function through:

• Mitochondrial dynamics studies:

  • Monitor SPBC3B9.04 localization during mitochondrial fission/fusion events

  • Assess protein abundance changes during mitochondrial biogenesis

  • Investigate potential relocalization during mitochondrial stress

• Metabolic adaptation research:

  • Track SPBC3B9.04 levels during shifts between fermentation and respiration

  • Correlate protein abundance with mitochondrial activity measurements

  • Investigate potential post-translational modifications under different metabolic states

• Mitochondrial quality control:

  • Examine SPBC3B9.04 protein turnover during mitophagy

  • Assess changes in localization during mitochondrial stress responses

  • Investigate potential role in protein methylation during quality control processes

• Comparative studies across growth conditions:

  • Compare protein levels and localization in glucose vs. glycerol media

  • Assess changes during nitrogen starvation or other stress conditions

  • Investigate cell cycle-dependent changes in mitochondrial methyltransferase activity

These applications build on established protocols for studying mitochondrial proteins in fission yeast, such as those used for analyzing mitochondrial protein import and processing .

How might SPBC3B9.04 antibodies contribute to understanding the methyltransferase's role in epigenetic regulation?

While SPBC3B9.04 is predicted to be a mitochondrial methyltransferase, investigating its potential involvement in epigenetic regulation could open new research avenues:

• Mitochondrial DNA (mtDNA) methylation studies:

  • Use SPBC3B9.04 antibodies in chromatin immunoprecipitation (ChIP) assays targeting mtDNA

  • Compare methylation patterns in wild-type versus SPBC3B9.04 deletion strains

  • Correlate SPBC3B9.04 binding sites with mtDNA gene expression levels

• Mitochondria-to-nucleus signaling:

  • Investigate whether SPBC3B9.04 shuttles between mitochondria and nucleus under specific conditions

  • Use fractionation followed by western blotting to track localization

  • Examine potential role in retrograde signaling pathways

• Non-histone protein methylation:

  • Identify mitochondrial protein substrates using proteomics approaches

  • Verify methylation state changes using specific antibodies against methylated residues

  • Correlate methylation patterns with SPBC3B9.04 levels and activity

• RNA methylation:

  • Investigate potential role in mitochondrial RNA methylation

  • Use RNA immunoprecipitation followed by sequencing (RIP-seq)

  • Compare methylation patterns with SPBC3B9.04 binding sites

These approaches connect to research on gene regulation in fission yeast, which exhibits features like centromeric and telomeric regions under tight regulatory control by constitutive heterochromatin .

How can SPBC3B9.04 antibodies be integrated into high-throughput screening approaches?

Integrating SPBC3B9.04 antibodies into high-throughput screening offers several possibilities:

• Automated immunofluorescence screening:

  • Adapt SPBC3B9.04 antibody staining for microplate format

  • Screen chemical libraries for compounds affecting localization or abundance

  • Implement machine learning for image analysis and phenotypic classification

• Protein array applications:

  • Use antibodies to detect SPBC3B9.04 binding partners on protein arrays

  • Develop arrays to screen for SPBC3B9.04 substrates

  • Implement reverse-phase protein arrays for quantitative measurements across conditions

• CRISPR screening validation:

  • Use SPBC3B9.04 antibodies to validate hits from CRISPR screens

  • Assess protein levels, localization, or modifications in genetic perturbation libraries

  • Combine with high-content imaging for multiparametric phenotypic analysis

• Flow cytometry-based screens:

  • Adapt protocols for intracellular staining in fixed/permeabilized cells

  • Screen for genetic or chemical modifiers of SPBC3B9.04 levels

  • Implement FACS sorting to isolate cells with altered expression patterns