SPBP23A10.06 Antibody

What is SPBP23A10.06 and why is it significant for research?

SPBP23A10.06 is an uncharacterized mitochondrial carrier protein found in Schizosaccharomyces pombe (fission yeast) . Its significance stems from its potential role in mitochondrial function and cellular metabolism. Fission yeast serves as an excellent model organism for studying fundamental cellular processes due to its defined cylindrical shape and well-characterized genetics . The protein is part of a genomic region that includes other functionally important proteins involved in processes such as cell separation and growth control . Research methodologies typically involve genetic manipulation of S. pombe strains, followed by phenotypic analysis using microscopy and biochemical assays to understand the protein's function.

How is SPBP23A10.06 structurally characterized?

SPBP23A10.06 is characterized as a mitochondrial carrier protein with a molecular weight of approximately 36,404 Da . While detailed structural information is limited, research approaches typically include:

• Sequence analysis and alignment with known mitochondrial carriers

• Prediction of transmembrane domains using bioinformatics tools

• Expression of recombinant protein for structural studies

• Use of antibodies for localization and interaction studies

For structural analysis, researchers often employ techniques such as crystallography or cryo-EM after protein purification, though no definitive 3D structure appears in the current literature for this specific protein.

What experimental systems are most suitable for studying SPBP23A10.06?

The most appropriate experimental systems include:

When studying SPBP23A10.06, researchers typically use native expression in S. pombe combined with epitope tagging (GFP, FLAG, TAP) to facilitate detection and purification . This approach preserves the physiological context while enabling molecular analysis.

What are the optimal conditions for using SPBP23A10.06 antibody in Western blot applications?

For optimal Western blot results when using SPBP23A10.06 antibody:

• Sample preparation:

  • Extract proteins from S. pombe cells using established lysis protocols

  • Include protease inhibitors (leupeptin, pepstatin, bestatin, aprotinin at 2 μg/ml, and PMSF at 1 mM)

  • Resolve proteins on 10% SDS-PAGE gels

• Antibody incubation:

  • Primary antibody dilutions typically range from 1:1000 to 1:5000

  • Consider using 5% BSA or milk in TBST for blocking and antibody dilution

  • Overnight incubation at 4°C generally provides optimal results

• Detection:

  • Use appropriate species-specific secondary antibodies

  • For chemiluminescence detection, ECL systems provide good sensitivity

  • For quantitative analysis, consider fluorescent secondary antibodies

Control experiments should include wild-type and deletion strains to confirm specificity, as demonstrated in similar studies of S. pombe proteins .

How can SPBP23A10.06 antibody be validated for immunoprecipitation experiments?

Validation of SPBP23A10.06 antibody for immunoprecipitation requires:

• Specificity testing:

  • Compare immunoprecipitation results from wild-type and SPBP23A10.06 deletion strains

  • Confirm the appropriate molecular weight band (36.4 kDa) is present only in wild-type samples

• Crosslinking optimization:

  • Test various crosslinking conditions if studying protein complexes (formaldehyde or DSP)

  • Compare native versus crosslinked conditions to identify stable interactions

• RNA-protein interactions:

  • If studying RNA-binding capabilities, include RNase inhibitors (100 U/ml Superase-In)

  • Consider native RNA immunoprecipitation protocols similar to those used for other S. pombe proteins

• Mass spectrometry validation:

  • Confirm pulled-down protein identity using LC-MS/MS analysis

  • Compare with proteomics databases for S. pombe to ensure correct identification

For RNA-protein interaction studies, researchers have successfully used similar approaches with other S. pombe proteins, where RNA was purified from immunoprecipitates for downstream analysis .

What are the appropriate controls when using SPBP23A10.06 antibody in immunofluorescence studies?

Essential controls for immunofluorescence studies include:

• Negative controls:

  • SPBP23A10.06 deletion strain (SPBP23A10.06Δ)

  • Primary antibody omission

  • Isotype control antibody

• Positive controls:

  • Known mitochondrial markers (e.g., using Mitotracker Green or antibodies against established mitochondrial proteins like Sdh2-GFP )

  • Double labeling with established organelle markers

• Specificity controls:

  • Peptide competition assay using the immunogen peptide

  • Signal comparison in cells with varied expression levels

• Technical controls:

  • Autofluorescence assessment

  • Fixed versus live cell comparison if applicable

Based on similar studies with mitochondrial proteins in S. pombe, researchers should expect mitochondrial localization patterns that can be verified by colocalization with established markers like Sdh2-GFP .

How does SPBP23A10.06 function in mitochondrial processes?

While specific functions of SPBP23A10.06 remain to be fully characterized, research approaches to understand its role include:

• Gene deletion phenotypic analysis:

  • Comparison of mitochondrial morphology using microscopy

  • Assessment of respiratory capacity using oxygen consumption measurements

  • Evaluation of mitochondrial membrane potential using fluorescent dyes

• Protein-protein interaction studies:

  • Immunoprecipitation followed by mass spectrometry to identify interaction partners

  • Yeast two-hybrid screening for direct interactors

  • Proximity labeling approaches (BioID or APEX) to map the local interactome

• Metabolic profiling:

  • Comparative metabolomics of wild-type versus mutant strains

  • Mitochondrial substrate utilization assays

  • Assessment of cellular response to metabolic stress conditions

Research on other mitochondrial carrier proteins in S. pombe has revealed roles in processes such as maintaining mitochondrial integrity during growth phases. For example, proteomic analysis identified mitochondrial protein decreases in certain mutants, suggesting regulated degradation mechanisms .

How do mutations in SPBP23A10.06 affect mitochondrial health and cellular metabolism?

Investigating the effects of SPBP23A10.06 mutations requires:

• Creation of mutation libraries:

  • Site-directed mutagenesis of conserved residues

  • Random mutagenesis approaches

  • CRISPR-based editing in S. pombe

• Functional assessment:

  • Growth rate analysis under various carbon sources

  • Mitochondrial membrane potential measurements

  • ROS production quantification

  • ATP synthesis capacity

• Proteomics approach:

  • Quantitative proteomics using techniques like emPAI (exponentially modified protein abundance index)

  • Analysis of mitochondrial protein abundance changes

  • Assessment of compensatory mechanisms

Research on related mitochondrial proteins suggests that loss of function can lead to significant decreases in mitochondrial protein levels and altered metabolic profiles. For example, in mts3-1 mutants, mitochondrial proteins like Sdh2-GFP showed time-dependent decreases at restrictive temperatures .

What role might SPBP23A10.06 play in stress response pathways?

Based on patterns observed with other mitochondrial proteins in S. pombe:

• Stress response analysis:

  • Gene expression changes under oxidative stress

  • Protein localization under nutrient limitation

  • Response to temperature shifts

• Cell cycle-dependent regulation:

  • Analysis of protein levels and localization throughout the cell cycle

  • Comparison of vegetative growth (VEG) versus G0 phase responses

  • Integration with cell division and separation mechanisms

• Regulatory pathway mapping:

  • Epistasis analysis with known stress response genes

  • Phosphoproteomics to identify regulatory modifications

  • Transcriptional response analysis

Studies with other S. pombe proteins show distinct responses during different growth phases. For example, some mitochondrial proteins show marked degradation specifically in G0 phase upon proteasomal inactivation, but not during vegetative growth .

How can SPBP23A10.06 antibody be utilized in chromatin immunoprecipitation studies?

For researchers investigating potential nuclear roles of SPBP23A10.06:

• Protocol optimization:

  • Crosslinking conditions (1% formaldehyde for 10-15 minutes is typical)

  • Sonication parameters to achieve 200-500bp DNA fragments

  • Antibody concentration titration

  • Pre-clearing strategies to reduce background

• Controls and validation:

  • Input DNA control

  • IgG negative control

  • Positive control using known DNA-binding proteins

  • qPCR validation of enriched regions before sequencing

• Data analysis:

  • Peak calling using appropriate algorithms

  • Motif analysis of enriched regions

  • Integration with transcriptomic data

  • Comparison with known chromatin states

While SPBP23A10.06 is annotated as a mitochondrial carrier, some proteins have dual localization. Studies of transcription factor cascades in S. pombe have established methodologies that could be applied if nuclear functions are suspected .

What approaches can be used to study potential interactions between SPBP23A10.06 and RNA-binding proteins?

Given the importance of RNA-binding proteins in S. pombe biology:

• Co-immunoprecipitation strategies:

  • Native RNA immunoprecipitation protocols

  • Include RNase inhibitors (100 U/ml Superase-In)

  • Validate RNA preservation with control transcripts

• RNA target identification:

  • RNA sequencing of co-precipitated RNA

  • RT-qPCR validation of selected targets

  • RNA-protein crosslinking methods (CLIP-seq)

• Functional validation:

  • Expression analysis of putative RNA targets in deletion strains

  • Secondary structure prediction of bound RNAs

  • Competition assays with synthetic RNA molecules

Research on RNA-binding proteins in S. pombe has revealed important regulatory mechanisms. For example, the Sts5 protein forms RNP granules that regulate cell polarity and growth through translational repression mechanisms .

How does the function of SPBP23A10.06 integrate with cell separation and morphogenesis pathways in S. pombe?

To investigate potential roles in cell morphology:

• Cell biology approaches:

  • Time-lapse microscopy of deletion mutants

  • Cell wall analysis using specific dyes

  • Quantitative morphology measurements

• Genetic interaction mapping:

  • Double mutant analysis with known morphogenesis genes

  • Suppressor screens to identify functional relationships

  • Epistasis tests with cell separation pathway components

• Specific pathway analysis:

  • Integration with Cdc42 GTPase regulation

  • Analysis of potential roles in the SIN pathway

  • Investigation of NDR kinase Orb6 interactions

S. pombe displays distinctive patterns of polarized growth and cell division that are regulated by various pathways. Research has shown that proteins in the SPBP23A10 genomic region may be involved in cell separation, as some encode cell wall hydrolytic enzymes necessary for this process .

What are common pitfalls when working with antibodies against low-abundance S. pombe proteins?

When working with potentially low-abundance proteins like SPBP23A10.06:

• Extraction optimization:

  • Use efficient cell disruption methods (e.g., bead beating with Zirconia beads)

  • Include protease inhibitor cocktails

  • Optimize buffer conditions for protein stability

• Signal enhancement strategies:

  • Consider using enhanced chemiluminescence systems

  • Implement signal amplification approaches

  • Increase exposure time while monitoring background

• Epitope access improvement:

  • Test different membrane types (PVDF vs. nitrocellulose)

  • Vary detergent types and concentrations

  • Consider antigen retrieval methods for fixed samples

• Specificity validation:

  • Use deletion strains as negative controls

  • Include competition assays with immunizing peptide

  • Compare results with epitope-tagged versions of the protein

Studies with other S. pombe proteins have shown that optimizing extraction conditions is critical, and the addition of glycerol (e.g., 50%) in storage buffers helps maintain antibody activity .

How can researchers optimize co-immunoprecipitation protocols for detecting transient interactions?

For capturing transient protein-protein interactions:

• Crosslinking approaches:

  • Optimize formaldehyde concentration (0.1-1%) and time

  • Test chemical crosslinkers with different spacer lengths

  • Consider photo-activatable crosslinkers for precise timing

• Buffer modifications:

  • Adjust salt concentration to preserve weak interactions

  • Test different detergent types and concentrations

  • Include stabilizing agents like glycerol or specific ions

• Rapid capture techniques:

  • Develop workflows minimizing time between lysis and capture

  • Consider on-bead digestion for mass spectrometry

  • Use proximity labeling approaches (BioID, APEX) as alternatives

• Validation strategies:

  • Reciprocal co-immunoprecipitations

  • Yeast two-hybrid or split-reporter confirmations

  • Localization studies to confirm spatial proximity

Researchers studying RNA-binding proteins in S. pombe have successfully used native conditions with appropriate RNase inhibitors to preserve complexes .

What analytical methods are most effective for quantifying changes in SPBP23A10.06 expression or localization?

For quantitative analysis:

• Expression quantification:

  • Western blot with fluorescent secondary antibodies

  • Mass spectrometry using techniques like emPAI

  • RT-qPCR for transcript level analysis

• Localization quantification:

  • Digital image analysis of immunofluorescence

  • Subcellular fractionation followed by Western blotting

  • Flow cytometry if using fluorescent protein fusions

• Dynamic analysis:

  • FRAP (Fluorescence Recovery After Photobleaching) for mobility

  • Time-lapse imaging for temporal patterns

  • Pulse-chase experiments for turnover rates

• Statistical approaches:

  • Use appropriate statistical tests (t-test, ANOVA)

  • Implement multiple biological and technical replicates

  • Consider normalization to housekeeping genes/proteins

For reliable quantification, research on mitochondrial proteins in S. pombe has employed both fluorescence microscopy with GFP-tagged proteins and proteomic approaches with mass spectrometry .

How can SPBP23A10.06 antibodies be used in studies comparing wild-type and mutant S. pombe strains?

When comparing different genetic backgrounds:

• Experimental design considerations:

  • Match growth conditions precisely

  • Harvest cells at equivalent growth phases

  • Process all samples in parallel

• Controls:

  • Include loading controls (e.g., actin, tubulin)

  • Use multiple independent mutant isolates

  • Consider complementation controls

• Quantification approaches:

  • Normalize to multiple reference proteins

  • Use digital imaging systems with linear range verification

  • Implement replicate analyses for statistical validation

• Phenotypic correlation:

  • Link protein expression/localization to cellular phenotypes

  • Perform rescue experiments with wild-type gene

  • Consider dose-response relationships if using regulatable promoters