SPAC1952.10c Antibody

What is SPAC1952.10c and what is its significance in S. pombe research?

SPAC1952.10c is a protein encoded by the genome of Schizosaccharomyces pombe (strain 972/ATCC 24843), commonly known as fission yeast. The specific function of this protein remains an area of active research, but as a component of the S. pombe proteome, it's valuable for studies of yeast cell biology, genetics, and comparative genomics. The protein corresponds to UniProt accession number Q9UUJ8 and is part of the broader research effort to characterize the complete S. pombe proteome .

To investigate this protein effectively, researchers need reliable detection methods that can specifically identify SPAC1952.10c in the context of other cellular proteins. This is where the SPAC1952.10c antibody provides significant value for research applications, allowing for specific detection and analysis of this protein in various experimental contexts.

What applications has the SPAC1952.10c antibody been validated for?

The SPAC1952.10c antibody has been specifically validated for:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Western blot (WB) for identification of the antigen

How should I properly store and handle this antibody to maintain its functionality?

For optimal preservation of antibody activity:

• Store the antibody at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles, which can degrade antibody performance

• The antibody is formulated in liquid form with a storage buffer containing:

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

  • 0.03% Proclin 300 as a preservative

When working with the antibody, allow it to equilibrate to room temperature before opening the vial, and always handle with clean, nuclease-free pipette tips to prevent contamination.

What is the recommended protocol for using SPAC1952.10c antibody in Western blot applications?

While the specific optimal dilution for this antibody must be determined empirically for each experimental system, the following general protocol is recommended:

• Sample Preparation:

  • Extract proteins from S. pombe using standard lysis buffers (RIPA or NP-40 based)

  • Quantify protein concentration using Bradford or BCA assay

  • Prepare samples in Laemmli buffer with DTT or β-mercaptoethanol

• Gel Electrophoresis and Transfer:

  • Separate 20-50 μg protein/lane on 10-12% SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane at 100V for 1 hour

• Immunoblotting:

  • Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with SPAC1952.10c antibody (starting dilution range: 1:500-1:2000) overnight at 4°C

  • Wash 3x with TBST, 5 minutes each

  • Incubate with HRP-conjugated secondary anti-rabbit antibody (1:5000) for 1 hour

  • Develop using ECL substrate and image

• Controls:

  • Include wild-type S. pombe extract as positive control

  • Consider using SPAC1952.10c knockout strain (if available) as negative control

Researchers should note that validation studies have shown that 20-30% of antibodies used in the literature may not recognize their intended targets, underscoring the importance of proper controls in Western blot applications .

What strategies can I use to validate the specificity of the SPAC1952.10c antibody?

Comprehensive validation is essential for confidence in experimental results. Consider these approaches:

• Genetic Validation:

  • Compare signals between wild-type and SPAC1952.10c deletion strains

  • Use strains with tagged versions of SPAC1952.10c (e.g., GFP-fusion) as positive controls

• Immunodepletion:

  • Pre-incubate the antibody with excess purified antigen before application

  • Signal reduction confirms specificity to the target antigen

• Cross-reactivity Assessment:

  • Test on related yeast species to evaluate cross-reactivity

  • Perform protein array experiments to assess binding to non-target proteins

• Orthogonal Methods:

  • Confirm key findings using independent techniques (mass spectrometry, RNA expression)

  • Consider using alternative antibodies targeting different epitopes of the same protein

Remember that antibody validation is application-specific—an antibody performing well in Western blot may not be suitable for immunohistochemistry or other applications .

How can I troubleshoot weak or absent signals when using the SPAC1952.10c antibody?

When experiencing detection issues with the SPAC1952.10c antibody, systematic troubleshooting can help identify and address the problem:

If problems persist after standard troubleshooting, consider that approximately 20-30% of commercial antibodies may not effectively recognize their intended targets according to large-scale validation studies .

What factors might affect the binding efficiency of this polyclonal antibody?

Several experimental and biological factors can influence antibody performance:

• Sample Preparation:

  • Denaturing conditions may disrupt epitopes recognized by the antibody

  • Different lysis buffers can affect protein conformation and epitope accessibility

• Protein Modifications:

  • Post-translational modifications may mask or create epitopes

  • Sample processing can introduce artificial modifications

• Environmental Factors:

  • Buffer pH and salt concentration affect antibody-antigen interactions

  • Detergents may influence epitope presentation and antibody binding

• Antibody Characteristics:

  • Being a polyclonal preparation, lot-to-lot variability may occur

  • Storage conditions and age of antibody affect performance

• Target Protein Properties:

  • Expression levels of SPAC1952.10c may vary under different conditions

  • Protein localization may affect extraction efficiency and detectability

Understanding these factors helps in designing robust experiments with appropriate controls .

How can I develop a quantitative assay using the SPAC1952.10c antibody?

Developing a quantitative assay requires careful calibration and validation:

• Standard Curve Development:

  • Generate recombinant SPAC1952.10c protein at known concentrations

  • Create standard curves using purified protein for each experiment

• ELISA Optimization:

  • Determine optimal antibody concentration through checkerboard titration

  • Establish linear range for quantification

  • Validate reproducibility across multiple runs

• Quantitative Western Blot:

  • Include known quantities of recombinant protein as standards

  • Use fluorescently-labeled secondary antibodies for wider linear range

  • Perform densitometry using appropriate software (ImageJ, etc.)

• Data Analysis:

  • Correct for background signal

  • Normalize to loading controls (e.g., actin, tubulin)

  • Apply appropriate statistical analyses for biological replicates

• Assay Validation Parameters:

  • Determine lower limit of detection (LLOD)

  • Assess intra-assay and inter-assay coefficients of variation

  • Evaluate specificity using knockout controls

This methodological approach draws on principles similar to those used in population pharmacokinetic modeling and antibody characterization studies .

How might I adapt immunoprecipitation protocols specifically for SPAC1952.10c in S. pombe?

Optimizing immunoprecipitation (IP) for SPAC1952.10c requires consideration of yeast-specific challenges:

• Cell Lysis Optimization:

  • S. pombe has a robust cell wall requiring effective disruption methods

  • Use glass beads lysis in cold buffer containing:

    • 50mM HEPES pH 7.5

    • 150mM NaCl

    • 1mM EDTA

    • 1% Triton X-100

    • Protease inhibitor cocktail

• Pre-clearing Strategy:

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

  • Use non-immune rabbit IgG as a negative control for non-specific binding

• Antibody Coupling:

  • For best results, covalently couple the SPAC1952.10c antibody to protein G-sepharose using dimethylpimelimidate (20mM)

  • This approach has proven successful in IP protocols for checkpoint complex isolation

• IP Conditions:

  • Incubate cleared lysate with antibody-coupled beads for 3-4 hours at 4°C with gentle rotation

  • Wash stringently (at least 3 times) with buffer containing 250-350mM NaCl

  • Elute bound proteins with SDS-PAGE sample buffer or gentle elution buffer

• Verification:

  • Analyze by Western blot using a portion of the SPAC1952.10c antibody

  • Consider mass spectrometry to identify interacting partners

This protocol adapts techniques used for other S. pombe protein complexes while accounting for the specific properties of SPAC1952.10c .

What considerations are important when using this antibody for studying protein-protein interactions?

When investigating SPAC1952.10c interactions, several methodological considerations are crucial:

• Preserving Native Interactions:

  • Use mild lysis conditions to maintain protein complexes

  • Consider crosslinking approaches for transient interactions

  • Optimize salt and detergent concentrations empirically

• Controls for Specificity:

  • Parallel IP with non-immune IgG is essential

  • Competition with recombinant antigen can verify specific interactions

  • Reverse IP with antibodies against suspected interacting partners

• Buffer Optimization:

  • Test different buffer compositions that maintain both antibody binding and protein-protein interactions

  • Consider specialized buffers that may better preserve interactions specific to yeast proteins

• Downstream Analysis:

  • Silver staining can reveal co-immunoprecipitated proteins

  • Mass spectrometry provides unbiased identification of interacting partners

  • Follow-up Western blotting confirms specific interactions

• Functional Validation:

  • Genetic approaches (double mutants, synthetic lethality)

  • In vitro binding assays with purified components

  • Structure-based predictions of interaction interfaces

These approaches have been successfully employed for characterizing protein complexes in S. pombe, such as DNA damage checkpoint complexes .

How does the polyclonal nature of this antibody compare with monoclonal antibodies for research applications?

The polyclonal nature of the SPAC1952.10c antibody offers distinct advantages and limitations compared to monoclonal alternatives:

For critical research applications, confirming findings with both antibody types may provide complementary data. Studies have shown that neutralizing monoclonal antibodies can retain effectiveness against viral variants, suggesting precise epitope targeting can be maintained even with genetic variation in the target .

What methods can be used to compare SPAC1952.10c function across different yeast species?

Comparative analysis of SPAC1952.10c across yeast species requires specialized approaches:

• Sequence-Based Comparison:

  • Identify orthologs through sequence alignment tools

  • Analyze sequence conservation using BLAST, SIM scores, and E-values

  • Examine domain architecture for functional conservation

• Cross-Species Antibody Validation:

  • Test SPAC1952.10c antibody against related proteins in other yeasts

  • Determine epitope conservation through sequence analysis

  • Use epitope mapping to identify cross-reactive regions

• Heterologous Expression Systems:

  • Express S. pombe SPAC1952.10c in S. cerevisiae to test functional complementation

  • Create chimeric proteins to identify functionally conserved domains

  • Use heterologous systems to identify species-specific interaction partners

• Comparative Functional Assays:

  • Perform parallel knockout/knockdown studies across species

  • Compare phenotypes under various stress conditions

  • Analyze growth parameters in defined media

• Structural Biology Approaches:

  • Compare 3D structures of orthologous proteins when available

  • Use homology modeling to predict structural conservation

  • Identify conserved surface patches as potential interaction sites

This multi-faceted approach can reveal evolutionary conservation and divergence of function, similar to methods used in antibody sequence analysis pipelines .

How might emerging antibody engineering technologies improve future studies of SPAC1952.10c?

Emerging technologies offer exciting possibilities for next-generation SPAC1952.10c research:

• Machine Learning Applications:

  • AI-based epitope prediction for optimized antibody design

  • Computational approaches like ASAP-SML (Antibody Sequence Analysis Pipeline using Statistical testing and Machine Learning) can identify features that distinguish effective antibodies

  • In silico modeling of antibody-antigen interactions for improved specificity

• Single-Domain Antibodies:

  • Development of nanobodies against SPAC1952.10c for applications requiring smaller probes

  • Enhanced penetration into subcellular compartments

  • Greater stability under various experimental conditions

• Recombinant Antibody Fragments:

  • Engineering Fab or scFv fragments for improved tissue penetration

  • Site-specific labeling for super-resolution microscopy

  • Multispecific formats for simultaneous targeting of interaction partners

• In-Cell Antibody Applications:

  • Intrabodies designed to track SPAC1952.10c in living cells

  • Antibody-mediated protein degradation (TRIM-Away technology)

  • Proximity labeling using antibody-enzyme fusions

• Integrated Multimodal Systems:

  • Combining antibody recognition with CRISPR-based genomic targeting

  • Antibody-oligonucleotide conjugates for spatial transcriptomics

  • Mass cytometry with metal-tagged antibodies for multiplexed detection

These advanced approaches build upon established antibody technologies while leveraging computational and bioengineering advances to create more precise research tools .

What role might the SPAC1952.10c antibody play in understanding broader cellular pathways in yeast?

The SPAC1952.10c antibody can serve as an important tool for elucidating broader cellular networks:

• Pathway Mapping Applications:

  • Identification of SPAC1952.10c in protein complexes via co-immunoprecipitation followed by mass spectrometry

  • Tracking protein redistribution during cell cycle progression

  • Monitoring changes in protein-protein interactions under stress conditions

• Systems Biology Integration:

  • Combining SPAC1952.10c localization data with transcriptomics and metabolomics

  • Network analysis to position SPAC1952.10c within broader cellular processes

  • Multi-omics studies incorporating antibody-based protein detection

• Evolutionary Conservation Analysis:

  • Comparative studies of orthologous proteins across fungal species

  • Identification of conserved interaction networks

  • Tracking evolutionary changes in protein function and localization

• Translational Research Potential:

  • Using yeast as a model for conserved cellular processes in higher eukaryotes

  • Identifying potential antifungal targets if SPAC1952.10c proves essential

  • Understanding fundamental biological processes through this model organism

• Methodological Advancements:

  • Development of yeast-specific proximity labeling approaches

  • Adaptation of semi-mechanistic population pharmacokinetic modeling approaches from mammalian systems

  • Creation of integrated preclinical/clinical models incorporating yeast data

This integrative approach parallels the semi-mechanistic modeling methods used for antibody pharmacokinetics research, adapted to the context of basic yeast cell biology .

What are the critical quality control parameters that should be assessed for each new lot of SPAC1952.10c antibody?

Consistent experimental results require rigorous quality control for each antibody lot:

• Affinity Assessment:

  • ELISA-based determination of binding constants

  • Surface Plasmon Resonance (SPR) for real-time binding kinetics

  • Competitive binding assays with established antibody lots

• Specificity Verification:

  • Western blot against S. pombe lysates (wild-type vs. knockout)

  • Protein microarray analysis to assess cross-reactivity

  • Immunoprecipitation followed by mass spectrometry

• Sensitivity Determination:

  • Limit of detection in typical applications

  • Signal-to-noise ratio compared to previous lots

  • Minimal detectable concentration of purified protein

• Functional Performance:

  • Side-by-side comparison with previous lots in standard assays

  • Application-specific validation (WB, ELISA, IP)

  • Performance consistency across different buffer conditions

• Stability Analysis:

  • Accelerated stability testing

  • Freeze-thaw tolerance assessment

  • Long-term storage stability monitoring

Research has shown that approximately 20-30% of antibodies used in literature don't effectively recognize their intended targets, highlighting the critical importance of comprehensive validation protocols .

How can I integrate computational approaches to better predict epitopes recognized by this polyclonal antibody?

Advanced computational methods can enhance understanding of antibody-antigen interactions:

• Epitope Prediction Algorithms:

  • B-cell epitope prediction tools (BepiPred, DiscoTope)

  • Structural epitope mapping using protein 3D models

  • Physicochemical property analysis (hydrophilicity, flexibility, accessibility)

• Molecular Dynamics Simulations:

  • Modeling antibody-antigen complexes

  • Simulating binding energetics

  • Predicting conformational epitopes

• Machine Learning Integration:

  • Training models on known antibody-antigen interactions

  • Feature extraction from antibody sequences

  • Applying pipelines like ASAP-SML that use statistical testing and machine learning

• Sequence-Structure Relationships:

  • Identify conserved structural motifs in antigens

  • Predict antibody binding based on CDR structure

  • Apply germline, CDR canonical structure, and isoelectric point analyses

• Cross-Reactivity Assessment:

  • Proteome-wide scanning for similar epitopes

  • Off-target binding prediction

  • Homology-based cross-reactivity modeling