SPBC887.17 Antibody

What is SPBC887.17 and what role does it play in fission yeast?

SPBC887.17 is a gene that encodes a uracil permease in Schizosaccharomyces pombe (fission yeast) . Uracil permeases are membrane proteins responsible for the uptake of uracil across the cell membrane, playing a crucial role in nucleotide metabolism. In S. pombe, this protein is particularly significant as it functions within the cellular transport mechanisms essential for nucleobase acquisition.

The protein has been identified with UniProt Number O94300 and Entrez Gene ID 2541241 . Understanding this protein's function is important for researchers studying membrane transport, nucleotide metabolism, and various cellular processes in fission yeast models.

What are the primary applications for SPBC887.17 Antibody in research settings?

SPBC887.17 Antibody has been validated for two primary research applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): Useful for quantitative detection of SPBC887.17 protein in complex samples .

• Western Blotting (WB): Enables researchers to identify and analyze SPBC887.17 protein expression levels and molecular weight in cell lysates .

These applications make the antibody particularly valuable for researchers investigating membrane transport mechanisms, protein-protein interactions, and gene expression regulation in fission yeast models. The antibody is designed with specificity for yeast species reactivity, making it ideal for S. pombe research .

How should researchers design optimal Western blotting protocols for SPBC887.17 detection?

When designing Western blotting protocols for SPBC887.17 detection, researchers should consider the following methodological approach:

• Sample Preparation:

  • Prepare total cell lysates using glass beads in lysis buffer containing 150 mM NaCl and 10 mM Tris-HCl (pH 7.0)

  • Add 0.5% Triton X-100 and 0.5% deoxycholate to the buffer

  • Include protease inhibitors: 0.4 mM phenylmethylsulfonyl fluoride and 1× protease inhibitor cocktail

• Gel Electrophoresis:

  • Load equal amounts of total protein onto a 15% polyacrylamide gel

  • Transfer proteins to nitrocellulose membranes

• Antibody Incubation:

  • Use the purified rabbit polyclonal SPBC887.17 Antibody at a recommended dilution (typically 1:1000-1:2000)

  • Consider using TAT-1 antibody against S. pombe tubulin as a loading control

• Controls:

  • Include the provided 200 μg antigens as a positive control

  • Use the provided 1 ml pre-immune serum as a negative control

• Storage and Handling:

  • Store antibody at -20°C or -80°C for optimal preservation of activity

What methodological approaches should be considered when incorporating SPBC887.17 Antibody in TSC pathway studies?

When investigating the TSC (Tuberous Sclerosis Complex) pathway using SPBC887.17 Antibody, researchers should consider these methodological approaches:

• Experimental Framework:

  • Design experiments that examine the relationship between SPBC887.17 (uracil permease) and components of the TSC pathway

  • Consider nitrogen starvation conditions, as the TSC pathway is involved in nutrient sensing

• Gene Expression Analysis:

  • Utilize Northern blotting to examine changes in SPBC887.17 expression under different conditions

  • When preparing RNA, follow established protocols (Jensen et al. 1983; Thomas et al. 1980)

  • Consider genome-wide expression profiling to contextualize SPBC887.17 within the broader response

• Subcellular Fractionation:

  • Prepare spheroplasts by incubating 10^10 cells at 37°C for 1 hour in spheroplasts buffer

  • Utilize fractionation to determine the subcellular localization of SPBC887.17 and its potential co-localization with TSC pathway components

• Genetic Interaction Studies:

  • Consider analyzing SPBC887.17 in tsc1-null or tsc2-null backgrounds

  • Look for effects on gene expression profiles during nitrogen starvation conditions

How can researchers validate the specificity of SPBC887.17 Antibody results?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For SPBC887.17 Antibody, consider these validation approaches:

• Genetic Controls:

  • Use SPBC887.17 deletion strains as negative controls

  • Employ SPBC887.17 overexpression systems as positive controls

  • Compare wild-type and mutant strains to confirm specificity

• Biochemical Validation:

  • Perform competitive binding assays using the provided 200 μg antigen (positive control)

  • Pre-absorb the antibody with purified antigen and verify signal reduction

  • Conduct peptide competition assays using synthetic peptides covering different regions of SPBC887.17

• Cross-reactivity Assessment:

  • Test the antibody against related permeases to confirm specificity

  • Examine reactivity in different species, considering the antibody is developed for yeast specificity

• Technical Controls:

  • Include the pre-immune serum provided with the antibody as a negative control

  • Use loading controls (e.g., anti-tubulin) for Western blotting experiments

  • Implement isotype controls to identify non-specific binding

What approaches are recommended for analyzing SPBC887.17 expression under nitrogen starvation conditions?

Nitrogen starvation significantly impacts gene expression in S. pombe, including genes encoding permeases . When analyzing SPBC887.17 expression under these conditions:

• Experimental Setup:

  • Culture cells in YES liquid medium to early-log phase

  • Split cultures into experimental (nitrogen-depleted EMM) and reference (standard medium) conditions

  • Maintain cultures for 3 hours before analysis

• Expression Analysis Methods:

  • Northern Blotting: Use PCR-amplified probes labeled with [α-32P]dCTP

  • RNA-Seq: For genome-wide expression profiling

  • Quantitative PCR: For targeted expression analysis

  • Microarray Analysis: Compare expression under experimental vs. reference conditions

• Data Analysis Approach:

  • Calculate fold changes in expression between nitrogen-starved and reference conditions

  • Compare wild-type responses to tsc1- or tsc2-null mutants

  • Apply statistical analysis to determine significance of expression changes

  • Consider time-course experiments to capture dynamic responses

• Protein-Level Verification:

  • Use SPBC887.17 Antibody in Western blotting to confirm that transcript-level changes translate to protein-level changes

  • Quantify protein levels relative to appropriate loading controls

How does SPBC887.17 function compare across different yeast species and what implications does this have for antibody cross-reactivity?

Understanding the evolutionary conservation of SPBC887.17 and its functional homologs provides important context for antibody applications:

• Functional Conservation:

  • SPBC887.17 encodes a uracil permease in S. pombe

  • Compare with similar permeases in Saccharomyces cerevisiae (budding yeast) and other fungal species

  • Note structural and functional differences that might affect antibody recognition

• Cross-Reactivity Analysis:

  • The SPBC887.17 Antibody is developed with "yeast" species reactivity

  • Consider testing cross-reactivity with related permeases in different yeast species

  • Sequence alignment of the immunogen region across species can predict potential cross-reactivity

• Evolutionary Implications:

  • Assess conservation of SPBC887.17 functional domains

  • Consider how variations in protein structure might impact antibody binding

  • Evaluate the potential for using this antibody in comparative studies across yeast species

What methodological approaches should be used when integrating SPBC887.17 Antibody data with genomic and transcriptomic datasets?

Integrating antibody-based protein detection with genomic and transcriptomic data requires carefully designed methodological approaches:

• Multi-omics Integration Strategy:

  • Correlate protein expression levels (detected with SPBC887.17 Antibody) with corresponding mRNA levels

  • Consider time-lag between transcription and translation when interpreting data

  • Look for discrepancies that might indicate post-transcriptional regulation

• Experimental Design for Integration:

  • Collect samples for protein and RNA analysis from the same experimental conditions

  • In nitrogen starvation studies, analyze both protein levels (using Western blotting) and mRNA levels (using Northern blotting)

  • Design time-course experiments to capture dynamic relationships

• Data Analysis Framework:

  • Apply normalization methods appropriate for each data type

  • Use statistical approaches that account for different noise characteristics

  • Consider machine learning approaches for identifying complex relationships

• Validation Approaches:

  • Confirm key findings using orthogonal techniques

  • Use genetic perturbations (e.g., SPBC887.17 overexpression or deletion) to validate functional relationships

  • Consider how changes in culture conditions affect correlation between protein and mRNA levels

What are the most common technical challenges when using SPBC887.17 Antibody and how can researchers address them?

When working with SPBC887.17 Antibody, researchers may encounter these technical challenges:

• High Background in Western Blots:

  • Cause: Insufficient blocking, contamination, or non-specific binding

  • Solution: Optimize blocking conditions (try 5% non-fat milk or BSA), increase washing steps, and titrate antibody concentration

  • Alternative: Use the provided pre-immune serum to identify non-specific binding patterns

• Weak or Absent Signal:

  • Cause: Insufficient protein, degraded antibody, or inefficient transfer

  • Solution: Increase protein loading, verify antibody storage conditions (-20°C or -80°C) , and optimize transfer parameters

  • Verification: Use the provided antigen as a positive control to confirm antibody activity

• Multiple Bands or Unexpected Band Size:

  • Cause: Post-translational modifications, degradation, or splice variants

  • Solution: Include protease inhibitors (0.4 mM phenylmethylsulfonyl fluoride and 1× protease inhibitor cocktail)

  • Validation: Compare with literature or perform additional experiments with tagged versions of SPBC887.17

• Poor Reproducibility:

  • Cause: Variations in experimental conditions or sample preparation

  • Solution: Standardize protocols, particularly cell lysis methods using glass beads in defined lysis buffer

  • Control: Include consistent positive and negative controls across experiments

How should researchers interpret conflicting results between SPBC887.17 protein expression and corresponding gene expression data?

When faced with discrepancies between protein levels (detected via SPBC887.17 Antibody) and gene expression data:

• Systematic Analysis Approach:

  • Compare time points carefully, as protein expression typically lags behind mRNA changes

  • Consider whether conflicting results occur under specific conditions (e.g., nitrogen starvation)

  • Verify results using alternative methods for both protein and mRNA detection

• Biological Interpretation Framework:

  • Consider post-transcriptional regulation mechanisms (mRNA stability, translation efficiency)

  • Evaluate post-translational modifications or protein degradation pathways

  • Assess protein localization changes that might affect detection but not total expression

• Technical Considerations:

  • Verify antibody specificity under the specific experimental conditions

  • Ensure appropriate normalization for both protein and RNA measurements

  • Consider sensitivity differences between protein and RNA detection methods

• Experimental Validation Strategies:

  • Design pulse-chase experiments to assess protein stability

  • Use translation inhibitors to distinguish transcriptional from post-transcriptional effects

  • Consider genetic approaches (e.g., mutations affecting mRNA processing or translation)

What emerging methodologies could enhance the utility of SPBC887.17 Antibody in membrane protein research?

Emerging methodologies that could advance SPBC887.17 research include:

• Advanced Imaging Techniques:

  • Super-resolution microscopy for precise localization of SPBC887.17 in yeast membranes

  • Live-cell imaging using fluorescently tagged secondary antibodies

  • Correlative light and electron microscopy to relate protein localization to membrane ultrastructure

• Proximity Labeling Approaches:

  • BioID or APEX2 proximity labeling to identify interaction partners of SPBC887.17

  • Combine with mass spectrometry for comprehensive interaction mapping

  • Integrate with antibody-based detection for validation of interactions

• Single-Cell Analysis:

  • Adaptation of SPBC887.17 Antibody protocols for single-cell protein quantification

  • Correlation of single-cell protein levels with transcriptomics data

  • Analysis of cell-to-cell variability in SPBC887.17 expression and localization

• Cryo-Electron Microscopy Applications:

  • Using SPBC887.17 Antibody for protein localization in cryo-preserved samples

  • Structural studies of SPBC887.17 in native membrane environments

  • Integration with tomography for 3D reconstruction of membrane transport complexes

How might SPBC887.17 Antibody contribute to understanding the relationship between nutrient sensing and membrane transport in eukaryotic systems?

SPBC887.17 Antibody offers potential for investigating broader biological questions:

• Nutrient Sensing and TSC Pathway Research:

  • Investigate how SPBC887.17 expression and localization respond to TSC pathway perturbations

  • Explore connections between uracil transport and TORC1 signaling

  • Study how nitrogen starvation affects SPBC887.17 membrane distribution

• Comparative Systems Biology Approaches:

  • Use SPBC887.17 Antibody to compare uracil permease regulation across fungal species

  • Investigate conservation of regulatory mechanisms between yeast permeases and human transporters

  • Develop systems biology models incorporating membrane transport and nutrient sensing

• Therapeutic Implications for Human Disease Models:

  • Apply insights from SPBC887.17 regulation to understand related human transporters

  • Explore connections to diseases involving nucleobase transport defects

  • Investigate potential relevance to tuberous sclerosis complex, leveraging the existing research on TSC pathway in fission yeast

• Methodological Advances for Membrane Protein Research:

  • Develop improved protocols for membrane protein extraction and analysis

  • Refine antibody-based approaches for studying dynamic changes in membrane protein localization

  • Integrate with lipidomics to understand membrane composition effects on transporter function