SPBC31F10.17c Antibody

What is SPBC31F10.17c and why is it significant for yeast research?

SPBC31F10.17c refers to a specific gene locus in Schizosaccharomyces pombe (fission yeast) encoding a protein cataloged under UniProt accession number P87318. The significance of this protein lies in its potential role in cellular processes unique to S. pombe. Antibodies against this protein enable researchers to study its expression, localization, and functional interactions through techniques such as western blotting, immunoprecipitation, and immunofluorescence microscopy. The study of SPBC31F10.17c contributes to our broader understanding of conserved eukaryotic cellular mechanisms, as S. pombe serves as an excellent model organism with many pathways conserved in higher eukaryotes including humans .

How should SPBC31F10.17c antibody be stored and handled to maintain optimal activity?

For optimal preservation of antibody activity, SPBC31F10.17c antibody should be stored following similar protocols to other research antibodies. Store the antibody at -20°C to -70°C for long-term storage (up to 12 months from date of receipt). For medium-term storage (up to 1 month), keep at 2-8°C under sterile conditions after reconstitution. For reconstituted antibodies, aliquot into single-use volumes to avoid repeated freeze-thaw cycles, which can significantly reduce activity. When handling, maintain sterile conditions and avoid contamination. Before each use, allow the antibody to equilibrate to room temperature and briefly centrifuge to collect solution at the bottom of the tube. Similar to other research antibodies, activity can be expected to remain stable for approximately 6 months at -20°C to -70°C under proper storage conditions .

What are the recommended applications for SPBC31F10.17c antibody?

Based on common antibody applications in yeast research, SPBC31F10.17c antibody can likely be utilized in multiple experimental techniques. Western blotting represents the primary application for detecting the target protein in cell lysates, with recommended dilutions typically between 1:500 to 1:2000 depending on antibody sensitivity and target abundance. Immunoprecipitation may be employed to isolate protein complexes containing SPBC31F10.17c, while immunofluorescence microscopy enables visualization of subcellular localization patterns. For flow cytometry applications, intracellular staining protocols would be necessary, potentially requiring cell fixation with paraformaldehyde and permeabilization with agents such as saponin. As with all antibodies, optimal dilutions should be determined empirically for each application and cell type. Researchers should validate specificity using appropriate controls including knockout/deletion strains when available .

How can I optimize western blot protocols specifically for SPBC31F10.17c detection in fission yeast extracts?

Optimizing western blot protocols for SPBC31F10.17c detection requires careful consideration of several parameters. For fission yeast protein extraction, use either mechanical disruption (glass beads) or enzymatic methods (zymolyase treatment followed by detergent lysis) under conditions that preserve protein integrity. Buffer systems should contain protease inhibitors and potentially phosphatase inhibitors if post-translational modifications are of interest.

For gel electrophoresis, 10-12% polyacrylamide gels typically provide optimal resolution for mid-sized proteins. During transfer, PVDF membranes often yield better results than nitrocellulose for yeast proteins. Begin antibody optimization with a dilution range test (1:500, 1:1000, 1:2000) in 5% non-fat milk or BSA in TBST. Overnight incubation at 4°C often improves specific binding while reducing background.

Critical controls should include:

• A negative control using pre-immune serum

• A competitive inhibition control using purified antigen

• Ideally, a SPBC31F10.17c deletion strain sample

If background remains problematic, try increasing blocking agent concentration (up to 10%), extending blocking time, adding 0.1-0.5% Tween-20 to wash buffers, or performing antibody pre-absorption with yeast extract from a SPBC31F10.17c deletion strain .

What are the recommended protocols for immunoprecipitation of SPBC31F10.17c-containing complexes?

For successful immunoprecipitation of SPBC31F10.17c-containing complexes, begin with optimized cell lysis conditions that preserve protein-protein interactions. A gentle lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40 or 0.5% Triton X-100, supplemented with protease inhibitors is recommended. For yeast cells, mechanical disruption using glass beads is often necessary to break the cell wall efficiently.

Pre-clear lysates by incubation with Protein A/G beads alone to reduce non-specific binding. For the immunoprecipitation, use 2-5 μg of SPBC31F10.17c antibody per 500 μg of total protein lysate. Incubate the antibody-lysate mixture at 4°C for 2-4 hours or overnight with gentle rotation. Add pre-washed Protein A/G beads and continue incubation for 1-2 hours. Wash the beads 4-5 times with lysis buffer containing reduced detergent concentration.

For analyzing protein complexes, elute bound proteins using either low pH glycine buffer or SDS sample buffer for subsequent SDS-PAGE analysis. For protein interaction studies, consider chemical crosslinking before lysis to stabilize transient interactions. Always include appropriate controls such as non-immune IgG and, if available, immunoprecipitation from SPBC31F10.17c deletion strains to identify non-specific interactions .

How can I validate the specificity of SPBC31F10.17c antibody for immunofluorescence applications?

Validating antibody specificity for immunofluorescence is critical for obtaining reliable localization data. Begin with a comprehensive validation approach using multiple controls. The gold standard validation requires parallel staining of wild-type and SPBC31F10.17c deletion strains. Absence of signal in the deletion strain strongly supports antibody specificity.

If gene deletion strains are unavailable, employ alternative validation strategies:

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before staining

• Correlation with fluorescent protein tagging: Compare antibody staining pattern with GFP-tagged SPBC31F10.17c

• RNA interference: Compare staining in cells with normal versus reduced SPBC31F10.17c expression

• Multiple antibodies: Use two antibodies recognizing different epitopes of SPBC31F10.17c

Optimize fixation conditions specifically for yeast cells, comparing formaldehyde (3-4%, 15-30 minutes) and methanol fixation (-20°C, 6-10 minutes) to determine which best preserves epitope accessibility. For S. pombe, enzymatic cell wall digestion with zymolyase or glucanases prior to fixation often improves antibody penetration.

Start with antibody dilutions between 1:50 to 1:200 and optimize based on signal-to-noise ratio. Include appropriate blocking steps (5-10% normal serum from the secondary antibody species) to minimize non-specific binding .

How can SPBC31F10.17c antibody be utilized in chromatin immunoprecipitation (ChIP) experiments?

Utilizing SPBC31F10.17c antibody for ChIP requires careful optimization due to the technical challenges of chromatin preparation from yeast cells. The protocol should begin with proper crosslinking using 1% formaldehyde for 10-15 minutes, followed by quenching with glycine. For S. pombe, cell wall digestion with zymolyase prior to mechanical lysis significantly improves chromatin extraction efficiency.

Chromatin shearing should be optimized to generate fragments between 200-500 bp, typically requiring 10-20 sonication cycles (30 seconds on/30 seconds off) on ice. For immunoprecipitation, use 3-5 μg of SPBC31F10.17c antibody per 25-50 μg of chromatin. Include appropriate controls:

• Input chromatin (10% of starting material)

• No-antibody control

• Non-specific IgG control

• Positive control using antibody against a known chromatin-associated protein

Prior to full-scale experiments, perform ChIP-qPCR targeting candidate regions to verify enrichment. For ChIP-seq applications, library preparation should include size selection for fragments in the 200-500 bp range and sequencing depth of at least 20 million reads per sample.

To address potential artifacts, consider performing biological replicates and validating key findings with complementary approaches such as CUT&RUN or using epitope-tagged versions of SPBC31F10.17c if the antibody shows limitations in chromatin immunoprecipitation efficiency .

What are the considerations for using SPBC31F10.17c antibody in co-immunoprecipitation coupled with mass spectrometry?

Implementing co-immunoprecipitation (co-IP) with mass spectrometry requires strategic planning to identify genuine SPBC31F10.17c interacting partners while minimizing false positives. Begin by optimizing lysis conditions that preserve protein-protein interactions while achieving efficient protein extraction from S. pombe. A buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 (or 0.5% NP-40), supplemented with protease inhibitors and phosphatase inhibitors, often provides a good starting point.

For the co-IP procedure, use sufficient antibody (5-10 μg per mg of lysate) and extend incubation times (overnight at 4°C) to capture weaker interactions. Wash conditions should balance removal of non-specific interactions while preserving genuine ones—typically 4-5 washes with decreasing detergent concentrations. For elution, avoid denaturing conditions if mass spectrometry compatibility is required; consider gentle elution using excess immunizing peptide or low pH glycine buffer followed by neutralization.

Critical controls must include:

• Parallel processing of samples from wild-type and SPBC31F10.17c deletion strains

• IgG control from the same species as the SPBC31F10.17c antibody

• Reciprocal co-IPs using antibodies against identified interactors

For mass spectrometry analysis, consider SILAC labeling of control and experimental samples to enable quantitative discrimination between specific and non-specific interactions. Filter mass spectrometry results using statistical thresholds (typically enrichment factor >2 and p-value <0.05) to identify high-confidence interactors .

Can SPBC31F10.17c antibody be used for super-resolution microscopy, and what protocol modifications are necessary?

SPBC31F10.17c antibody can indeed be adapted for super-resolution microscopy techniques such as STORM, PALM, or SIM, but several critical modifications to standard immunofluorescence protocols are necessary. For optimal results in super-resolution imaging of yeast cells:

• Sample preparation requires meticulous attention:

  • Use thinner coverslips (No. 1.5H, 170 ± 5 μm) for improved optical performance

  • For STORM/PALM, cells must be immobilized on poly-L-lysine coated coverslips

  • Cell wall digestion should be carefully optimized using zymolyase to improve antibody accessibility without compromising cellular structures

• Fixation and permeabilization protocols need modification:

  • Use freshly prepared 3% paraformaldehyde with 0.1% glutaraldehyde for better ultrastructural preservation

  • Consider shorter fixation times (10-15 minutes) to preserve antigenicity

  • Use detergent concentrations below 0.2% to minimize structural disruption

• Antibody labeling considerations:

  • Higher antibody concentrations (1:50 to 1:100) may be necessary

  • Extended incubation times (overnight at 4°C) improve labeling density

  • For STORM/PALM, use secondary antibodies conjugated to photoswitchable fluorophores like Alexa Fluor 647 or Cy5

• For specific super-resolution techniques:

  • STORM: Imaging buffer must contain an oxygen scavenging system (glucose oxidase/catalase) and thiol compounds (MEA or β-mercaptoethanol)

  • SIM: Higher laser powers are needed, requiring careful testing for photobleaching

  • PALM: Consider photo-convertible fluorescent protein tagging as an alternative if antibody labeling density is insufficient

The resolution improvement (typically 20-120 nm depending on the technique) enables visualization of SPBC31F10.17c distribution at previously unattainable detail, potentially revealing discrete subcellular structures or protein clustering not visible with conventional microscopy .

How can I address weak or absent signals when using SPBC31F10.17c antibody in western blotting?

Weak or absent signals when using SPBC31F10.17c antibody in western blotting may result from multiple factors that require systematic troubleshooting. Begin by verifying protein extraction efficiency through total protein staining (Ponceau S or similar) of your membrane. For S. pombe proteins, extraction requires efficient cell wall disruption—ensure your protocol employs appropriate mechanical (glass beads) or enzymatic (zymolyase) methods.

If extraction appears successful, focus on antibody-related parameters:

• Try decreasing antibody dilution (1:250 instead of 1:1000)

• Extend primary antibody incubation to overnight at 4°C

• Ensure antibody hasn't degraded by checking expiration date and storage conditions

• Consider trying a different lot of the antibody

For detection-related issues:

• Increase ECL substrate exposure time or switch to more sensitive detection reagents

• For low-abundance proteins, consider using signal amplification systems

• Check secondary antibody compatibility and freshness

If the protein is difficult to detect due to poor transfer, modify your transfer protocol:

• Extend transfer time or increase voltage (while monitoring heat)

• For hydrophobic proteins, consider adding 0.1% SDS to transfer buffer

• Try PVDF membrane instead of nitrocellulose for better protein retention

The most common cause of complete signal absence is incorrect target identification or expression. Verify that SPBC31F10.17c is expressed under your experimental conditions by checking expression databases or RT-PCR. Consider using a positive control sample where the protein is known to be highly expressed or using tagged recombinant SPBC31F10.17c as a standard .

What are the common causes of non-specific binding when using SPBC31F10.17c antibody, and how can they be mitigated?

Non-specific binding represents a frequent challenge when working with antibodies in yeast systems. For SPBC31F10.17c antibody, several common causes and mitigation strategies include:

• Insufficient blocking:

  • Increase blocking agent concentration from 5% to 10%

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Try alternative blocking agents (BSA, casein, or commercial blockers) if milk protein is problematic

• Cross-reactivity with similar epitopes:

  • Pre-absorb antibody with yeast lysate from SPBC31F10.17c deletion strain

  • Increase washing stringency by adding salt (up to 500 mM NaCl) to wash buffers

  • Consider affinity purification of the antibody against the specific immunizing peptide

• Detergent-related issues:

  • Optimize Tween-20 concentration in wash buffers (typically 0.05-0.1%)

  • For immunofluorescence, reduce detergent concentration during permeabilization

  • For problematic samples, add 0.1% Triton X-100 to antibody dilution buffer

• Secondary antibody problems:

  • Use highly cross-adsorbed secondary antibodies to reduce species cross-reactivity

  • Dilute secondary antibodies more extensively (1:5000 to 1:10000)

  • Include 1-5% serum from the host species of the secondary antibody in its dilution buffer

• Sample-specific interference:

  • For western blots, consider using gradient gels to better separate proteins

  • For immunoprecipitation, increase pre-clearing steps with beads alone

  • For immunofluorescence, include an autofluorescence quenching step

The most definitive way to identify true non-specific binding is by comparing staining patterns between wild-type and SPBC31F10.17c deletion strains. Signals present in both samples represent non-specific binding that requires further optimization .

How can I quantitatively assess SPBC31F10.17c antibody specificity and sensitivity?

Quantitative assessment of antibody specificity and sensitivity requires systematic evaluation using multiple complementary approaches. Begin with a dot blot titration assay using purified recombinant SPBC31F10.17c protein (if available) at concentrations ranging from 1 ng to 1 μg, alongside potential cross-reactive proteins. Calculate the limit of detection and dynamic range from the resulting signal intensities.

For western blot applications, perform a dilution series of total yeast lysate (6.25 μg to 100 μg) and plot band intensity versus protein amount to determine linearity range and sensitivity. Compare signal between wild-type and SPBC31F10.17c deletion/knockdown strains to calculate a specificity index (SI):

SI = Signal from wild-type / Signal from deletion strain

A high SI (>10) indicates excellent specificity, while values <2 suggest problematic cross-reactivity.

For immunofluorescence applications, quantify the signal-to-background ratio in multiple cellular compartments using digital image analysis. Compare mean fluorescence intensity between specific cellular locations and background regions, with ratios >5 typically indicating acceptable specificity.

To evaluate batch-to-batch consistency, create a reference sample (preferably a stable cell line or standardized yeast extract) and store aliquots at -80°C. Test each new antibody lot against this standard and calculate a consistency ratio:

Consistency Ratio = Signal from new lot / Signal from reference lot

Values between 0.8-1.2 indicate acceptable consistency, while deviations beyond this range warrant adjustment of working dilutions or consideration of alternative lots .

How can SPBC31F10.17c antibody be employed in studying protein-protein interactions during the cell cycle in S. pombe?

Investigating cell cycle-dependent protein-protein interactions involving SPBC31F10.17c requires sophisticated experimental design combining synchronization techniques with immunoprecipitation approaches. Begin by establishing a synchronization protocol using one of these methods:

• Temperature-sensitive cdc mutants

• Hydroxyurea arrest-release

• Lactose gradient centrifugation

• Nitrogen starvation and release

Verify synchronization efficiency by flow cytometry or microscopic examination of septation index. Collect samples at defined time points spanning the entire cell cycle (typically 6-8 time points).

For each time point, perform co-immunoprecipitation using SPBC31F10.17c antibody under conditions that preserve physiological interactions. Native lysis buffers containing 0.1-0.5% NP-40 or digitonin often work well for preserving cell cycle-specific complexes. Consider dual crosslinking approaches using DSP (dithiobis[succinimidylpropionate]) followed by formaldehyde to capture transient interactions.

Analyze immunoprecipitates using:

• Western blotting for known or candidate interactors

• Mass spectrometry for unbiased interaction discovery

For quantitative assessment of dynamic interactions, implement SILAC or TMT labeling strategies to compare interaction stoichiometry across cell cycle stages. Alternatively, proximity labeling approaches using BioID or APEX2 fused to SPBC31F10.17c can capture neighboring proteins in living cells at different cell cycle stages.

Validate key interactions using complementary approaches:

• Fluorescence microscopy to confirm co-localization

• FRET/FLIM to measure direct interaction

• Yeast two-hybrid assays to confirm direct binding

• Reciprocal co-IPs using antibodies against identified partners

This multi-dimensional approach can reveal how SPBC31F10.17c interaction networks reorganize during cell cycle progression, potentially identifying regulatory mechanisms that control its function .

What methodological approaches can address potential epitope masking in different cellular compartments when using SPBC31F10.17c antibody?

Epitope masking represents a significant challenge when studying proteins across different cellular compartments, potentially leading to false-negative results in certain locations. For SPBC31F10.17c antibody, several methodological approaches can address this issue:

• Multiple fixation and epitope retrieval strategies:

  • Compare crosslinking fixatives (formaldehyde, DSP) with precipitating fixatives (methanol, acetone)

  • Implement epitope retrieval techniques including:

    • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0)

    • Enzymatic retrieval using proteases like proteinase K (1-5 μg/ml for 5-10 minutes)

    • Detergent-based permeabilization series (0.1-1% Triton X-100, 0.1-0.5% SDS)

• Alternative antibody approaches:

  • Use multiple antibodies recognizing different epitopes of SPBC31F10.17c

  • Compare N-terminal versus C-terminal targeting antibodies

  • Consider generating a peptide antibody against a known accessible region

• Complementary visualization strategies:

  • Compare antibody-based detection with fluorescent protein tagging

  • Implement proximity labeling techniques (BioID, APEX2) which function independently of epitope accessibility

  • Use RNA detection methods (RNA FISH) to localize SPBC31F10.17c mRNA as a proxy for protein localization

• Comprehensive sub-cellular fractionation:

  • Isolate distinct cellular compartments (nucleus, cytoplasm, membrane, etc.)

  • Analyze each fraction by western blotting under different denaturing conditions

  • Compare detection efficiency with increasing detergent or chaotropic agent concentrations

• Quantitative assessment framework:

  • Create a comparative accessibility index by normalizing antibody signal to that of an epitope-tagged version

  • Generate accessibility maps across cellular compartments under different experimental conditions

  • Document condition-specific detection protocols for future reference

By systematically implementing these approaches, researchers can develop compartment-specific protocols for SPBC31F10.17c detection, ensuring comprehensive visualization across all cellular locations .

How can SPBC31F10.17c antibody be used in conjunction with CRISPR-Cas9 genome editing to study protein function?

Integrating SPBC31F10.17c antibody with CRISPR-Cas9 genome editing enables powerful approaches for studying protein function through precise genetic manipulation and subsequent phenotypic analysis. This combined strategy offers several advanced research applications:

• Validation of CRISPR-mediated modifications:

  • Use the antibody to confirm successful gene knockout by western blotting

  • Verify epitope tag insertion by comparing native and tagged protein detection

  • Assess efficiency of conditional degron systems through quantitative western blotting

• Domain-specific function analysis:

  • Generate domain deletion mutants using CRISPR and analyze expression/localization with the antibody

  • Create point mutations in putative functional domains and assess effects on protein stability

  • Engineer domain swaps with related proteins and use domain-specific antibodies to track chimeric proteins

• Protein regulation studies:

  • Implement CRISPR interference (CRISPRi) to achieve tunable repression and correlate expression levels with function

  • Engineer promoter replacements for controlled expression and use the antibody for quantitative analysis

  • Create allelic series with graduated phenotypes and correlate with protein expression levels

• Interaction partner validation:

  • Perform IP-MS with SPBC31F10.17c antibody before and after CRISPR deletion of suspected interaction partners

  • Create specific interaction-disrupting mutations based on structural predictions

  • Engineer proximity-based tagging systems and compare with native interaction detection

• Implementation protocol:

  Stage	CRISPR Application	Antibody Utilization
  Design	gRNA selection for target modification	Epitope accessibility analysis for tag placement
  Validation	Genotyping and sequencing	Expression confirmation by western blot
  Functional analysis	Phenotypic assays	Protein localization by IF, complex formation by IP
  Mechanistic studies	Secondary modifications	Quantitative analysis of protein dynamics

By systematically combining CRISPR-based genetic engineering with antibody-based protein analysis, researchers can achieve unprecedented insights into SPBC31F10.17c function, regulation, and interaction networks. This integrated approach bridges the gap between genetic manipulation and biochemical characterization, enabling comprehensive functional studies in the native cellular context .

What are the most critical considerations when selecting SPBC31F10.17c antibody for specific applications?

Selecting the appropriate SPBC31F10.17c antibody for specific applications requires careful evaluation of several critical factors to ensure experimental success. First, consider the validation status of the antibody for your particular application. An antibody with demonstrated performance in western blotting may not necessarily work in immunoprecipitation or immunofluorescence. Review available validation data, particularly those demonstrating specificity using SPBC31F10.17c deletion strains or knockdown approaches.

Second, evaluate the epitope recognition characteristics. Antibodies recognizing different regions of SPBC31F10.17c may perform differently under various experimental conditions. N-terminal antibodies may be preferable for detecting processed forms, while C-terminal antibodies often work better for proteins with conserved functional domains. For proteins with multiple isoforms or post-translational modifications, select antibodies that can discriminate between these forms if relevant to your research question.

Third, consider clone type and production method. Monoclonal antibodies offer high specificity and lot-to-lot consistency but may be sensitive to epitope changes. Polyclonal antibodies typically recognize multiple epitopes, providing robust detection but potentially introducing higher background and batch variation. For challenging applications like ChIP or IP-MS, high-affinity antibodies with demonstrated performance in these applications should be prioritized.

Fourth, match antibody characteristics to application requirements. For western blotting, antibodies with high affinity under denaturing conditions are ideal. For immunofluorescence, antibodies that recognize native conformations are preferred. For techniques requiring quantitative analysis, antibodies with demonstrated linear response ranges should be selected.

Finally, consider implementing alternative detection strategies (epitope tagging, fluorescent protein fusions) as complementary approaches when antibody limitations are encountered, particularly for low-abundance proteins or those with problematic detection characteristics .

How might future technological developments enhance the application scope of SPBC31F10.17c antibody in fission yeast research?

Emerging technologies are poised to significantly expand the application scope of antibodies like SPBC31F10.17c in fission yeast research. Single-cell proteomics approaches will enable analysis of protein expression heterogeneity within yeast populations, particularly valuable for studying transitions like sporulation or stress responses. Antibody-oligonucleotide conjugates will enable highly multiplexed protein detection through technologies like CODEX or MERFISH, allowing simultaneous visualization of dozens of proteins within the same sample.

In vivo protein dynamics will be revolutionized through techniques like optogenetic degrons combined with antibody-based quantification, enabling precise temporal control of protein levels followed by accurate measurement. For structural biology, advances in cryo-electron tomography combined with in-cell labeling using nanobody derivatives of existing antibodies will reveal native protein complex architectures at near-atomic resolution.

Spatial proteomics will benefit from emerging Spatial-seq technologies that combine antibody-based protein detection with transcriptomic analysis in the same sample, revealing protein-RNA relationships with subcellular resolution. For interaction studies, proximity-dependent RNA labeling methods like APEX-seq will complement traditional antibody-based co-IP approaches by identifying RNAs associated with SPBC31F10.17c in living cells.

The development of synthetic antibodies with enhanced properties—particularly those selected for proper folding in the cytosol—will enable new applications like intrabody expression for real-time tracking of native, untagged SPBC31F10.17c in living yeast. Finally, machine learning approaches will enhance experimental design by predicting optimal conditions for antibody applications based on protein sequence characteristics and previously successful protocols.