SPAC1002.16c Antibody

What is SPAC1002.16c and why is it important in fission yeast research?

SPAC1002.16c is a gene in Schizosaccharomyces pombe (fission yeast) that encodes a predicted nicotinic acid plasma membrane transporter. Understanding its function is important for several reasons:

• As a membrane transporter, it plays a potential role in nutrient acquisition pathways

• It represents one of many genes with specific roles in the fission yeast model system

• The protein's function may provide insights into conserved membrane transport mechanisms across species

According to genetic database information, SPAC1002.16c is categorized within the GO (Gene Ontology) classifications for specific biological processes, molecular functions, and cellular components .

What characteristics should researchers consider when selecting a SPAC1002.16c antibody?

When selecting a SPAC1002.16c antibody, researchers should evaluate:

• Antibody type: The commercially available SPAC1002.16c antibody is a polyclonal antibody raised in rabbit against recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) SPAC1002.16c protein .

• Validation status: Ensure the antibody has been validated for your specific application (e.g., Western blot, ELISA).

• Species reactivity: Confirm specificity for S. pombe, as antibody cross-reactivity with other yeast species may complicate data interpretation.

• Purification method: The available antibody is antigen affinity-purified, which enhances specificity .

• Storage conditions: Proper storage at -20°C or -80°C is essential to maintain antibody integrity and performance .

How does antibody validation differ between standard proteins and less-studied proteins like SPAC1002.16c?

Validation for well-characterized proteins often relies on established protocols and reference data. For less-studied proteins like SPAC1002.16c, validation requires additional considerations:

• Knockout controls: Generation of SPAC1002.16c knockout strains is essential for validating antibody specificity, similar to approaches used in validating other yeast protein antibodies .

• Orthogonal methods: Correlation with alternative detection methods (e.g., mass spectrometry, RNA-seq) provides stronger validation.

• Expression systems: Recombinant expression systems can help establish baseline signals for validation.

• Cross-reactivity assessment: Thorough testing against related membrane transporters is critical to confirm specificity.

Research shows well-characterized antibody reagents play a key role in research reproducibility, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays .

What is the optimal protocol for using SPAC1002.16c antibody in Western blot applications?

For optimal Western blot results with SPAC1002.16c antibody:

• Sample preparation:

  • Extract membrane proteins from S. pombe using appropriate buffer systems containing detergents suitable for membrane proteins

  • Include protease inhibitors to prevent degradation

  • For membrane proteins like SPAC1002.16c, avoid boiling samples as this may cause aggregation

• Gel electrophoresis and transfer:

  • Use 4-20% gradient gels for optimal separation

  • Transfer to nitrocellulose membranes at controlled voltage (e.g., 100V for 1 hour)

• Blocking and antibody incubation:

  • Block with 5% milk in TBST for 1 hour

  • Incubate with SPAC1002.16c antibody at recommended dilution overnight at 4°C

  • Wash thoroughly with TBST (3 times, 5 minutes each)

  • Incubate with appropriate secondary antibody for 1 hour at room temperature

• Detection and validation:

  • Use enhanced chemiluminescence for detection

  • Include positive and negative controls

  • Perform replicate experiments to ensure reproducibility

This protocol is based on standardized approaches for membrane protein detection in yeast .

How should researchers optimize immunoprecipitation experiments using SPAC1002.16c antibody?

For successful immunoprecipitation of SPAC1002.16c:

• Antibody-bead preparation:

  • Add 2 μg of SPAC1002.16c antibody to 500 μL of IP lysis buffer

  • Add 30 μL of Protein A Dynabeads (for rabbit antibodies)

  • Incubate with rocking for ~1 hour at 4°C

  • Wash twice to remove unbound antibodies

• Sample preparation:

  • Prepare membrane protein extracts from S. pombe using gentle lysis conditions

  • Adjust protein concentration to 1.5-2.0 mg/mL

  • Add protease inhibitor cocktail

• Immunoprecipitation:

  • Incubate prepared extract with antibody-bead conjugate for ~1 hour at 4°C

  • Collect unbound fractions

  • Wash beads three times with 1.0 mL of IP lysis buffer

  • Process for SDS-PAGE and Western blot analysis

• Detection specificity:

  • Use VeriBlot or similar secondary detection systems (0.3 μg/mL) to minimize detection of immunoprecipitating antibody heavy and light chains

This protocol is adapted from standardized approaches described for immunoprecipitation of membrane proteins .

What controls are necessary to ensure reliable results when working with SPAC1002.16c antibody?

Essential controls include:

• Genetic controls:

  • SPAC1002.16c knockout strains as negative controls

  • Strains overexpressing SPAC1002.16c as positive controls

  • Wild-type strains for baseline expression

• Antibody controls:

  • Non-specific IgG from the same species (rabbit) as a negative control

  • Pre-absorption with antigen to confirm specificity

  • Serial dilution of antibody to establish optimal concentration

• Technical controls:

  • Loading controls for Western blot (e.g., actin or tubulin)

  • Dye-free gel lanes to detect autofluorescence

  • Replicate experiments to assess reproducibility

• Validation controls:

  • Secondary antibody-only samples to detect non-specific binding

  • Correlation with orthogonal methods (e.g., mass spectrometry)

Research demonstrates that routine implementation of standardized antibody validation and reporting in immunoassays promotes improved reproducibility across the global life sciences community .

How can SPAC1002.16c antibody be utilized in studies of membrane transporter localization and dynamics?

The antibody can be strategically employed to investigate:

• Subcellular localization:

  • Immunofluorescence microscopy to determine precise membrane localization

  • Co-localization studies with other membrane markers

  • Changes in localization under different nutrient conditions, especially varying nicotinic acid concentrations

• Temporal dynamics:

  • Time-course experiments during cell cycle progression

  • Expression changes during sporulation or stress response

  • Correlation with RNA-binding proteins like Meu5 that regulate expression of membrane proteins

• Protein-protein interactions:

  • Co-immunoprecipitation to identify interaction partners

  • Proximity labeling approaches combined with mass spectrometry

  • Investigation of potential interactions with chromatin remodeling complexes like H2A.z

• Post-translational modifications:

  • Detection of phosphorylation, ubiquitination, or other modifications

  • Correlation of modifications with transporter activity

This multifaceted approach aligns with contemporary research on membrane protein dynamics in fission yeast .

What methodological approaches enable integration of SPAC1002.16c antibody data with functional genomics datasets?

Integration strategies include:

• Multi-omics correlation:

  • Correlation of protein expression data with transcriptomics

  • Integration with metabolomics data to link transporter function with metabolic changes

  • Comparison with proteomics datasets from different growth conditions

• Network analysis:

  • Construction of protein-protein interaction networks

  • Pathway analysis incorporating SPAC1002.16c function

  • Comparative analysis with other membrane transporters

• Systems biology approaches:

  • Mathematical modeling of transporter dynamics

  • Integration with genome-scale metabolic models

  • Simulation of transporter activity under varying conditions

• Comparative genomics:

  • Analysis across yeast species to identify conserved functions

  • Evolutionary studies of membrane transporter families

This integrated approach aligns with current fungal systems biology strategies described in research literature .

How might SPAC1002.16c antibody contribute to understanding the role of membrane transporters in stress response?

The antibody can reveal critical insights through:

• Stress-induced expression changes:

  • Quantification of protein levels under various stressors (oxidative, osmotic, nutritional)

  • Correlation with stress-responsive transcription factors

  • Comparison with other nutrient transporters during stress

• Functional analysis:

  • Association with stress-induced membrane reorganization

  • Changes in localization during stress response

  • Potential role in stress recovery

• Genetic interaction studies:

  • Combining with mutations in stress response pathways

  • Analysis in deletion backgrounds of stress-responsive genes

  • Investigation of genetic interactions with cyc1 and other stress-responsive genes

• Comparative analysis with stress-related proteins:

  • Co-regulation patterns with known stress proteins

  • Integration with data from stress response studies

Fission yeast stress response research reveals that membrane proteins often play critical roles in adaptation to changing environmental conditions .

What strategies can resolve common technical challenges when working with SPAC1002.16c antibody in Western blots?

Practical strategies for antibody validation address specificity, selectivity, and reproducibility in Western blot analysis, with emphasis on complementary methods to confirm observed effects .

How should quantitative analysis of SPAC1002.16c expression be approached using antibody-based methods?

For robust quantitative analysis:

• Sample standardization:

  • Normalize protein loading using multiple housekeeping proteins

  • Include calibration curves with recombinant SPAC1002.16c protein

  • Process all comparative samples simultaneously

• Image acquisition:

  • Use calibrated digital imaging systems with linear dynamic range

  • Capture multiple exposures to ensure signal is within linear range

  • Avoid saturated pixels that prevent accurate quantification

• Data analysis:

  • Apply background subtraction consistently across all samples

  • Use software that enables precise band intensity measurement

  • Analyze replicate samples to establish statistical significance

• Validation approaches:

  • Compare results from multiple antibody dilutions

  • Correlate Western blot data with other quantitative methods

  • Perform statistical analysis with appropriate tests (ANOVA, t-test)

Methodology papers emphasize that antibody should produce reproducible results within and between Western blotting experiments, with observed effects confirmed using complementary methods .

What approaches help distinguish genuine SPAC1002.16c signals from artifacts in immunolocalization experiments?

To differentiate true signals from artifacts:

• Rigorous controls:

  • Use SPAC1002.16c knockout cells as negative controls

  • Pre-absorb antibody with recombinant antigen

  • Include secondary antibody-only controls

• Validation techniques:

  • Confirm localization with multiple antibodies (if available)

  • Correlate with fluorescently tagged SPAC1002.16c

  • Use orthogonal approaches (subcellular fractionation)

• Image acquisition and analysis:

  • Use identical acquisition settings for experimental and control samples

  • Perform colocalization analysis with known membrane markers

  • Implement statistical analysis of signal distribution

• Signal verification:

  • Test specificity through competitive blocking with recombinant protein

  • Perform signal quantification across multiple cells and experiments

  • Apply deconvolution or super-resolution techniques for precise localization

Research demonstrates that standardized approaches to immunolocalization significantly improve reproducibility and reliability of results .

How might SPAC1002.16c antibody contribute to understanding membrane transporter evolution across fungal species?

Future evolutionary studies could leverage the antibody to:

• Comparative expression analysis:

  • Examine cross-reactivity with homologous transporters in related yeast species

  • Compare expression patterns and regulation across evolutionary distances

  • Investigate structural conservation of epitopes recognized by the antibody

• Functional conservation studies:

  • Determine if localization patterns are conserved across species

  • Investigate whether regulatory mechanisms are maintained through evolution

  • Study complementation of function across species

• Structural biology applications:

  • Use antibodies to purify native protein for structural studies

  • Compare membrane topology across species

  • Identify conserved functional domains

• Evolutionary proteomics:

  • Track evolutionary changes in post-translational modifications

  • Investigate co-evolution with interacting proteins

  • Study species-specific adaptations in membrane transporter function

Genome evolution studies in Hemiascomycete yeasts provide context for understanding the evolution of membrane transporters across fungal species .

What potential roles might SPAC1002.16c play in fission yeast sporulation and how could the antibody elucidate these functions?

The antibody could reveal sporulation-related functions through:

• Expression dynamics during sporulation:

  • Time-course analysis of protein levels throughout sporulation

  • Localization changes during forespore membrane formation

  • Potential roles in nutrient transport during spore maturation

• Functional investigations:

  • Examination of SPAC1002.16c in ΔSPAC1002.16c sporulation phenotypes

  • Comparison with other membrane proteins involved in sporulation

  • Investigation of potential interactions with Meu5 or other RNA-binding proteins

• Mechanistic studies:

  • Potential involvement in forespore membrane breakdown

  • Investigation of interactions with cytochrome c and other sporulation factors

  • Assessment of roles in spore wall formation

• Genetic interaction analysis:

  • Examination of genetic interactions with known sporulation genes

  • Construction of double mutants to identify synthetic phenotypes

  • Integration with sporulation-specific gene expression data

Research in S. pombe has identified that membrane transporters and RNA-binding proteins like Meu5 play crucial roles in sporulation, particularly in forespore membrane dynamics .

How could SPAC1002.16c antibody be integrated into functional genomics approaches to study membrane transporter networks?

Integration strategies include:

• Systematic interaction mapping:

  • Large-scale co-immunoprecipitation studies to map the interactome

  • Proximity labeling approaches to identify membrane-proximal proteins

  • Correlation of interactions with functional genomics data

• Advanced screening approaches:

  • Antibody-based screens in deletion libraries

  • Synthetic genetic array analysis incorporating antibody-based readouts

  • Chemical-genetic profiling with antibody detection

• Multi-dimensional data integration:

  • Correlation of protein expression with transcriptomics and metabolomics

  • Integration with protein-protein interaction networks

  • Incorporation into genome-scale metabolic models

• Systems-level analysis:

  • Network analysis of membrane transporter systems

  • Pathway modeling incorporating transporter functions

  • Mathematical modeling of nutrient acquisition systems

Current functional genomics studies in S. pombe demonstrate that integrative approaches provide comprehensive understanding of biological systems at the cellular level .