SPCC1494.07 Antibody

What is SPCC1494.07 and why is it relevant for research?

SPCC1494.07 is a gene identified in the fission yeast Schizosaccharomyces pombe genome. It appears in genomic studies as one of the genes that show significant expression changes in certain conditions. Specifically, it has been identified as a putative target of Upf1, a key factor in the nonsense-mediated mRNA decay (NMD) pathway . Additionally, SPCC1494.07 shows a notable decrease in expression (ratio of -1.085) in aneuploid strains containing the minichromosome Ch16 compared to normal haploid strains . Research on SPCC1494.07 and antibodies against its protein product contributes to our understanding of gene expression regulation in yeast, particularly in the context of RNA surveillance mechanisms and chromosomal abnormalities.

What are the known characteristics of the SPCC1494.07 protein?

While the search results do not provide specific details about the SPCC1494.07 protein structure or function, its presence in the list of Upf1 targets suggests it may be regulated post-transcriptionally through nonsense-mediated decay pathways . The protein likely has functional relevance in cellular processes affected by aneuploidy, as evidenced by its differential expression in cells containing the minichromosome Ch16 . Researchers investigating this protein would need to characterize its molecular weight, cellular localization, and functional domains to develop effective antibodies and experimental protocols.

How are antibodies against S. pombe proteins typically produced?

Antibodies against S. pombe proteins, including those like SPCC1494.07, are typically produced using one of two main approaches:

• Recombinant protein expression: The target gene (such as SPCC1494.07) is cloned and expressed in a heterologous system, commonly Escherichia coli with a tag for purification (e.g., His-tag), similar to the method described for Rhb1 antibody production . The purified protein is then used to immunize animals for polyclonal antibody production.

• Synthetic peptide approach: Short, unique peptide sequences from the target protein are synthesized and conjugated to carrier proteins before immunization.

For successful antibody production against SPCC1494.07, researchers should identify unique epitopes within the protein sequence that do not share significant homology with other S. pombe proteins to ensure specificity.

What are the recommended protocols for using SPCC1494.07 antibodies in Western blotting?

When using antibodies against SPCC1494.07 in Western blotting, researchers should follow these methodological considerations:

• Sample preparation: Total cell extracts from S. pombe can be prepared using methods similar to those described for Upf1:TAP protein detection . This typically involves mechanical cell disruption followed by extraction in an appropriate buffer containing protease inhibitors.

• Protein separation: Standard SDS-PAGE protocols are suitable, with gel percentage selected based on the predicted molecular weight of SPCC1494.07.

• Transfer and blocking: After transferring to a membrane, blocking with 3-5% non-fat milk or BSA in TBST is recommended.

• Antibody incubation: Primary antibody dilutions should be optimized (typically starting at 1:500-1:2000) and incubated overnight at 4°C. Secondary antibody selection should match the host species of the primary antibody.

• Detection: Enhanced chemiluminescence (ECL) systems are commonly used for detection.

• Controls: Include a positive control (overexpressed SPCC1494.07) and a negative control (extract from a SPCC1494.07 deletion strain) to validate antibody specificity.

How can SPCC1494.07 antibodies be used in chromatin immunoprecipitation (ChIP) studies?

For ChIP experiments using SPCC1494.07 antibodies, researchers should adapt protocols similar to those used for Swi6 ChIP in S. pombe :

• Crosslinking: Fix exponentially growing S. pombe cells (approximately 5×10^8 cells) with 3% formaldehyde for 30 minutes at 18°C, followed by glycine quenching (final concentration 0.125 M).

• Cell lysis and sonication: Wash cells with cold PBS, lyse with appropriate buffers, and sonicate to fragment chromatin (optimally 200-500 bp fragments).

• Immunoprecipitation: Incubate cleared lysates with SPCC1494.07 antibody for 4 hours at 4°C, followed by capture with Protein A/G beads.

• Washing and elution: Perform stringent washes to remove non-specific binding, then elute protein-DNA complexes.

• Reverse crosslinking and DNA purification: Reverse formaldehyde crosslinks and purify DNA for downstream analysis.

• Analysis: Quantify enrichment using qPCR, microarray, or sequencing approaches.

This approach would be particularly valuable for investigating whether SPCC1494.07 interacts with chromatin and identifying its potential binding sites across the genome.

What considerations are important for immunofluorescence microscopy with SPCC1494.07 antibodies?

For immunofluorescence microscopy using SPCC1494.07 antibodies in S. pombe:

• Cell fixation: Fix cells with either 3% paraformaldehyde (for structural preservation) or cold methanol (for better antibody accessibility to certain epitopes).

• Cell wall digestion: Treat with zymolyase or lysing enzymes to create spheroplasts, facilitating antibody penetration.

• Blocking: Use BSA or normal serum from the secondary antibody host species to prevent non-specific binding.

• Antibody incubation: Apply optimized dilutions of SPCC1494.07 primary antibody (starting at 1:100-1:500) and fluorophore-conjugated secondary antibody.

• Counterstaining: Include DAPI for nuclear visualization and other markers for subcellular compartments as needed.

• Controls: Parallel staining of SPCC1494.07 deletion strains is essential to confirm signal specificity.

• Image acquisition: Use appropriate filter sets and exposure settings to capture specific signals while minimizing background.

This technique would help determine the subcellular localization of SPCC1494.07 protein and potential changes in localization under different conditions or genetic backgrounds.

How can SPCC1494.07 antibodies be used to investigate nonsense-mediated decay mechanisms?

Given that SPCC1494.07 appears to be a putative target of Upf1, a key factor in nonsense-mediated decay (NMD) , antibodies against this protein can provide valuable insights into NMD mechanisms:

• Protein stability analysis: Compare SPCC1494.07 protein levels in wild-type versus upf1Δ, upf2Δ, and upf3Δ strains using Western blotting with SPCC1494.07 antibodies to determine if NMD affects not only mRNA but also protein abundance.

• Immunoprecipitation coupled with RNA analysis: Perform RNA immunoprecipitation (RIP) using SPCC1494.07 antibodies, followed by RT-qPCR or sequencing to identify associated RNAs, potentially revealing interactions with other NMD targets or NMD machinery components.

• Pulse-chase experiments: Combine metabolic labeling with immunoprecipitation using SPCC1494.07 antibodies to determine protein half-life in different genetic backgrounds, providing insights into post-translational regulation.

• Co-immunoprecipitation studies: Use SPCC1494.07 antibodies to pull down the protein and identify interacting partners by mass spectrometry, potentially revealing connections to the NMD machinery or other RNA processing pathways.

These approaches would add to our understanding of how NMD affects gene expression beyond mRNA degradation, potentially revealing feedback mechanisms or secondary effects of NMD deficiency.

How does aneuploidy affect SPCC1494.07 expression and what insights can antibody-based studies provide?

SPCC1494.07 shows decreased expression in aneuploid strains containing Ch16 , suggesting it is sensitive to chromosomal imbalances. Antibody-based studies can explore this phenomenon:

• Protein level quantification: Compare SPCC1494.07 protein levels across different aneuploid strains using Western blotting to determine if protein abundance correlates with mRNA changes or if post-translational compensation occurs.

• Chromatin association studies: Perform ChIP with antibodies against chromatin modifiers in the SPCC1494.07 locus in normal versus aneuploid cells to investigate epigenetic changes that might explain expression differences.

• Protein localization: Use immunofluorescence to examine whether aneuploidy affects SPCC1494.07 protein subcellular localization, potentially revealing stress responses or adaptive mechanisms.

• Protein modification analysis: Immunoprecipitate SPCC1494.07 from normal and aneuploid cells, then analyze post-translational modifications by mass spectrometry to identify regulatory changes.

These studies would contribute to understanding how cells respond to chromosomal imbalances at the protein level, potentially identifying compensatory mechanisms that maintain cellular homeostasis despite genomic instability.

What approaches can be used to validate the specificity of SPCC1494.07 antibodies?

Rigorous validation of SPCC1494.07 antibodies is critical for reliable research results. Comprehensive validation should include:

• Genetic validation: Compare antibody signal in wild-type versus SPCC1494.07 deletion strains across multiple techniques (Western blot, immunofluorescence, ChIP). A specific antibody should show signal only in wild-type cells.

• Overexpression validation: Test antibody against samples with endogenous versus overexpressed SPCC1494.07, confirming proportional signal increase with overexpression.

• Peptide competition assay: Pre-incubate antibody with the immunizing peptide or recombinant protein before application to samples; a specific antibody will show diminished signal.

• Immunoprecipitation-mass spectrometry: Perform immunoprecipitation followed by mass spectrometry to confirm the antibody pulls down SPCC1494.07 rather than cross-reactive proteins.

• Cross-species reactivity analysis: Test antibody against related proteins from other species to evaluate epitope specificity.

• Multiple antibody validation: Compare results using antibodies raised against different epitopes of the same protein to confirm consistency.

What are common troubleshooting strategies for weak or non-specific signals with SPCC1494.07 antibodies?

When encountering problems with SPCC1494.07 antibody performance, researchers should consider these methodological adjustments:

• For weak signals:

  • Increase antibody concentration incrementally

  • Extend primary antibody incubation time (overnight at 4°C)

  • Enhance detection sensitivity using amplification systems (e.g., biotin-streptavidin)

  • Optimize protein extraction methods to preserve epitopes

  • Increase protein loading amount for Western blots

• For non-specific signals:

  • Increase stringency of washing steps (higher salt concentration, longer washes)

  • Optimize blocking conditions (try different blocking agents: milk, BSA, normal serum)

  • Decrease antibody concentration

  • Pre-absorb antibody with cell lysate from knockout strains

  • Use more specific secondary antibodies with minimal cross-reactivity

• For high background:

  • Extend blocking time

  • Add detergent (0.1-0.3% Triton X-100) to antibody dilution buffer

  • Filter antibody solutions before use

  • Ensure all buffers are freshly prepared

These optimizations should be implemented systematically, changing one parameter at a time to identify the most effective conditions for SPCC1494.07 antibody applications.

How can researchers optimize immunoprecipitation protocols for SPCC1494.07 protein complex studies?

For successful immunoprecipitation of SPCC1494.07 and associated proteins:

• Lysis buffer optimization:

  • Test different detergent types and concentrations (NP-40, Triton X-100, CHAPS)

  • Adjust salt concentration to preserve interactions (typically 100-150mM NaCl for stable complexes; lower for weak interactions)

  • Include appropriate protease and phosphatase inhibitors

  • Consider crosslinking agents for transient interactions

• Antibody coupling strategies:

  • Compare direct immunoprecipitation versus pre-binding antibodies to beads

  • Test different antibody-to-bead ratios to maximize capture efficiency

  • Consider covalent coupling to beads to eliminate antibody contamination in eluted samples

• Washing conditions:

  • Develop a washing gradient with increasing stringency to determine optimal conditions

  • Include control washes to monitor non-specific binding reduction

• Elution methods:

  • Compare different elution strategies (low pH, high pH, competitive elution with peptides, SDS)

  • For mass spectrometry applications, use MS-compatible elution buffers

• Controls:

  • Include isotype-matched control antibodies

  • Perform parallel IPs from SPCC1494.07 deletion strains

  • Consider using tagged versions of SPCC1494.07 for parallel validation

These optimizations would enhance the specificity and yield of SPCC1494.07 protein complexes, enabling more comprehensive interactome studies.

What factors should be considered when interpreting quantitative differences in SPCC1494.07 levels across experimental conditions?

When analyzing quantitative changes in SPCC1494.07 levels using antibody-based methods:

• Technical considerations:

  • Ensure equal protein loading using multiple loading controls

  • Account for transfer efficiency variations in Western blots (using stain-free gels or total protein normalization)

  • Establish antibody linear dynamic range to ensure measurements fall within it

  • Use technical replicates to assess method variability

• Biological considerations:

  • Confirm changes at both mRNA and protein levels to distinguish transcriptional from post-transcriptional effects

  • Consider protein half-life when interpreting rapid changes

  • Evaluate cell cycle stage and growth phase effects on expression

  • Assess whether observed changes correlate with functional outcomes

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Use multiple biological replicates (minimum n=3) for meaningful comparisons

  • Implement power analysis to determine necessary sample sizes for detecting expected differences

  • Consider effect size in addition to statistical significance

• Validation approaches:

  • Confirm findings using orthogonal methods (e.g., mass spectrometry)

  • Demonstrate similar trends with different antibodies against the same protein

  • Use genetic approaches (overexpression, deletion) to verify antibody-based findings

These considerations help ensure that observed changes in SPCC1494.07 levels represent true biological phenomena rather than technical artifacts.

How can SPCC1494.07 antibody data be integrated with other -omics datasets?

Integration of antibody-based SPCC1494.07 data with other -omics approaches provides comprehensive insights:

• Transcriptome integration:

  • Compare protein levels (Western blot/immunofluorescence) with mRNA expression (microarray/RNA-seq) to identify post-transcriptional regulation

  • Correlate changes in SPCC1494.07 protein with global transcriptional responses

  • Analyze relationship between SPCC1494.07 and Upf1-regulated transcripts

• Proteome integration:

  • Compare immunoprecipitation results with global proteomics data to identify unique versus common interactors

  • Correlate SPCC1494.07 abundance changes with proteome-wide alterations in response to stress or genetic perturbations

  • Use antibody-based fractionation to enrich for SPCC1494.07-associated complexes prior to mass spectrometry

• Epigenome integration:

  • Compare ChIP-seq data from SPCC1494.07 antibodies with histone modification patterns and chromatin accessibility

  • Correlate SPCC1494.07 binding sites with transcriptional activity and Swi6 distribution patterns

• Network analysis:

  • Position SPCC1494.07 within protein-protein interaction networks using antibody-derived interactome data

  • Integrate with genetic interaction networks to identify functional relationships

  • Use protein association data in pathway enrichment analyses

For effective integration, researchers should standardize experimental conditions across platforms, implement robust normalization methods, and apply appropriate statistical approaches for multi-dimensional data analysis.

What comparative studies can be performed using SPCC1494.07 antibodies across different yeast species?

Cross-species studies using SPCC1494.07 antibodies can reveal evolutionary conservation and functional adaptation:

• Epitope conservation analysis:

  • Test SPCC1494.07 antibodies against homologous proteins in related yeasts (S. cerevisiae, S. japonicus, other Schizosaccharomyces species)

  • Map recognized epitopes across species to identify conserved functional domains

• Comparative expression studies:

  • Analyze protein expression patterns across species under standardized conditions

  • Compare subcellular localization of homologous proteins using immunofluorescence

  • Examine expression changes in response to stresses across species

• Functional conservation assessment:

  • Compare protein complex composition across species using immunoprecipitation

  • Analyze post-translational modifications of homologs using immunoprecipitation followed by mass spectrometry

  • Examine chromatin association patterns of homologous proteins using ChIP

• Complementation experiments:

  • Express homologs from different species in S. pombe SPCC1494.07 deletion strains

  • Use antibodies to confirm expression and localization of heterologous proteins

  • Correlate expression levels with functional complementation

These comparative approaches would provide insights into the evolutionary history of SPCC1494.07 and potentially reveal species-specific adaptations in its function or regulation.

How might SPCC1494.07 antibodies contribute to understanding stress response mechanisms in yeast?

Given SPCC1494.07's differential expression in aneuploid conditions, which represent a form of cellular stress , antibodies against this protein could be valuable tools for investigating stress responses:

• Stress-induced expression dynamics:

  • Monitor SPCC1494.07 protein levels under various stresses (oxidative, heat shock, nutrient limitation) using Western blotting

  • Compare protein stability and turnover rates under normal versus stress conditions

  • Examine stress-induced changes in post-translational modifications using immunoprecipitation and mass spectrometry

• Stress granule association:

  • Use immunofluorescence to determine if SPCC1494.07 localizes to stress granules or P-bodies during stress

  • Perform co-localization studies with known stress response factors

  • Analyze dynamics of association/dissociation during stress onset and recovery

• Chromatin reorganization:

  • Apply ChIP-seq to map changes in SPCC1494.07 genomic binding sites under stress conditions

  • Correlate binding patterns with transcriptional changes in stress response genes

  • Investigate interactions with chromatin remodeling factors during stress adaptation

• Protein interaction dynamics:

  • Compare SPCC1494.07 interactome under normal versus stress conditions

  • Identify stress-specific protein associations that might indicate role in adaptive responses

  • Analyze how these interactions are affected in mutants defective in stress signaling pathways

These studies would contribute to our understanding of cellular adaptation mechanisms and potentially reveal new functions for SPCC1494.07 in stress response pathways.

What potential therapeutic applications might arise from research using SPCC1494.07 antibodies?

While direct therapeutic applications from yeast research may seem distant, fundamental discoveries often translate to clinical relevance:

• Conserved pathway insights:

  • If SPCC1494.07 functions in evolutionarily conserved pathways (like NMD), antibody-based studies could reveal mechanisms relevant to human disease

  • Research on nonsense-mediated decay modulation could inform therapeutic approaches for genetic diseases caused by premature termination codons

• Drug screening applications:

  • Antibodies against SPCC1494.07 could be used to develop assays for screening compounds that affect NMD efficiency

  • Changes in protein levels or modifications could serve as readouts for drug effects on these pathways

• Biomarker development:

  • If human homologs show similar regulation patterns, insights from yeast could inform development of diagnostic or prognostic markers

  • Understanding protein behavior under aneuploidy could contribute to cancer research, where chromosomal abnormalities are common

• Synthetic biology applications:

  • Knowledge of SPCC1494.07 function could inform engineering of yeast strains with enhanced properties for biotechnology

  • Antibodies would serve as tools to validate engineered strains and monitor protein expression

While these translational aspects represent long-term potential outcomes, they highlight how fundamental research using tools like SPCC1494.07 antibodies contributes to the pipeline for therapeutic innovation.