SPAC17G8.11c Antibody

What is SPAC17G8.11c and why is it studied in Schizosaccharomyces pombe research?

SPAC17G8.11c is a gene locus in the fission yeast Schizosaccharomyces pombe (strain 972 / ATCC 24843). It represents one of many systematically named genomic elements in the comprehensive annotation of the S. pombe genome. This gene is of interest in fundamental fungal biology research, particularly for researchers studying conserved eukaryotic cellular mechanisms. S. pombe is a valuable model organism due to its cellular organization and genetic tractability, making SPAC17G8.11c potentially important for understanding conserved cellular processes .

What types of SPAC17G8.11c antibodies are currently available for research?

The primary type of SPAC17G8.11c antibody available is a polyclonal antibody raised in rabbit against recombinant Schizosaccharomyces pombe (strain 972 / ATCC 24843) SPAC17G8.11c protein. These antibodies are typically supplied in liquid form, containing preservation buffer (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4), and are purified using antigen affinity methods. The antibodies are designed for applications such as ELISA and Western Blot, with specific validation for these techniques .

What are the recommended storage conditions for SPAC17G8.11c antibodies?

For optimal stability and activity, SPAC17G8.11c antibodies should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and performance. The antibodies are typically supplied in a stabilizing buffer containing 50% glycerol and 0.03% Proclin 300 as a preservative, which helps maintain antibody integrity during storage. For working solutions, storage at 2-8°C for short periods is acceptable, but long-term storage should be at freezing temperatures .

How should researchers validate SPAC17G8.11c antibodies for specific applications?

Validation of SPAC17G8.11c antibodies should follow a multi-step approach:

• Positive and negative controls: Use wild-type S. pombe expressing SPAC17G8.11c as a positive control and knockout strains or heterologous systems without SPAC17G8.11c expression as negative controls.

• Titration experiments: Perform serial dilutions (typically starting from 1:100 to 1:10,000) to determine optimal working concentration for each application (Western blot, ELISA, immunohistochemistry).

• Cross-reactivity testing: Evaluate potential cross-reactivity with related proteins, particularly if working with multiple Schizosaccharomyces species or other fungi.

• Application-specific validation:

  • For Western blots: Verify single band of expected molecular weight

  • For ELISA: Establish standard curves using known quantities of recombinant protein

  • For immunostaining: Compare subcellular localization patterns with published data or GFP-fusion studies

What are the recommended protocols for using SPAC17G8.11c antibodies in Western blot applications?

For optimal Western blot results with SPAC17G8.11c antibodies:

• Sample preparation:

  • Lyse S. pombe cells using glass bead disruption in appropriate buffer (typically containing protease inhibitors)

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE gels

  • Transfer to PVDF or nitrocellulose membranes at 100V for 1 hour or 30V overnight

• Blocking and antibody incubation:

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

  • Incubate with SPAC17G8.11c antibody (start with 1:1000 dilution) overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with HRP-conjugated anti-rabbit secondary antibody for 1 hour at room temperature

• Detection:

  • Visualize using enhanced chemiluminescence reagents

  • Expected molecular weight should be verified against database information for the target protein

• Controls:

  • Include lysates from knockout strains as negative controls

  • Consider using tagged versions of the protein as positive controls

What considerations are important when designing immunoprecipitation experiments with SPAC17G8.11c antibodies?

When designing immunoprecipitation (IP) experiments with SPAC17G8.11c antibodies, researchers should consider:

• Lysis conditions:

  • Use non-denaturing buffers to preserve protein-protein interactions

  • Include appropriate protease and phosphatase inhibitors

  • Optimize salt concentration based on expected interaction strength

• Pre-clearing step:

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Match the species of Protein A/G beads to the antibody host (Protein A for rabbit polyclonal antibodies)

• Antibody amounts:

  • Titrate antibody amounts (typically 1-5 μg per mg of total protein)

  • Consider using cross-linking methods to prevent antibody co-elution

• Controls:

  • Include isotype control antibodies to identify non-specific interactions

  • Use knockout strains as negative controls

  • Consider including RNase/DNase treatment if investigating nucleic acid-mediated interactions

• Elution strategies:

  • If pursuing mass spectrometry, consider native elution with competing peptides

  • For standard applications, elution with reducing SDS sample buffer is usually sufficient

How can SPAC17G8.11c antibodies be used in comparative studies across different yeast species?

For cross-species comparative studies using SPAC17G8.11c antibodies:

• Sequence homology analysis:

  • Begin with bioinformatic analysis to identify sequence homologs in target species (e.g., Saccharomyces cerevisiae, Aspergillus flavus)

  • Align sequences to identify conserved epitope regions that might cross-react with the antibody

  • Calculate percent identity and similarity in antigenic regions

• Cross-reactivity validation:

  • Test antibody recognition using recombinant proteins or lysates from multiple species

  • Perform Western blots with samples from all species in parallel

  • Include appropriate controls for each species

• Epitope mapping approaches:

  • If cross-reactivity issues arise, consider epitope mapping to identify specific recognition sites

  • Design blocking peptides based on divergent regions to improve specificity

• Data interpretation considerations:

  • Account for differences in protein expression levels between species

  • Consider evolutionary conservation patterns when interpreting functional similarities

  • Validate key findings with species-specific antibodies when possible

What approaches can be used to study SPAC17G8.11c protein interactions and complexes?

Advanced approaches for studying SPAC17G8.11c protein interactions include:

• Co-immunoprecipitation (Co-IP) with tandem mass spectrometry:

  • Use SPAC17G8.11c antibody for IP followed by LC-MS/MS analysis

  • Compare results from wild-type and knockout strains to identify specific interactors

  • Validate key interactions with reciprocal Co-IPs using antibodies against identified partners

• Proximity labeling approaches:

  • Express SPAC17G8.11c fused to BioID or APEX2 enzymes

  • Identify proximal proteins through biotinylation and streptavidin pulldown

  • Use antibodies to validate proximity labeling results

• Crosslinking mass spectrometry (XL-MS):

  • Apply chemical crosslinkers to stabilize transient interactions

  • Perform IP with SPAC17G8.11c antibody

  • Identify crosslinked peptides by mass spectrometry to map interaction interfaces

• FRET or BiFC imaging:

  • Combine fluorescent protein fusions with antibody-based validation

  • Use SPAC17G8.11c antibodies for immunofluorescence to confirm localization patterns

  • Correlate interaction data with functional assays

What strategies can address epitope masking issues when using SPAC17G8.11c antibodies in fixed specimens?

To overcome epitope masking issues in fixed specimens:

• Optimization of fixation protocols:

  • Compare different fixatives (formaldehyde, methanol, acetone) for optimal epitope preservation

  • Test varying fixation durations (10 minutes to 24 hours) to minimize overfixation

  • Consider combining fixatives (e.g., formaldehyde followed by methanol) for dual benefits

• Antigen retrieval methods:

  • Heat-induced epitope retrieval: Test buffers at different pH values (citrate buffer pH 6.0, Tris-EDTA pH 9.0)

  • Enzymatic digestion: Try proteinase K, trypsin, or pepsin at various concentrations

  • Develop specialized protocols for yeast cell wall penetration (e.g., zymolyase treatment)

• Signal amplification techniques:

  • Implement tyramide signal amplification for low-abundance targets

  • Use polymer-based detection systems for enhanced sensitivity

  • Consider quantum dot conjugates for improved signal-to-noise ratio

• Microwave-assisted immunostaining:

  • Apply microwave treatment during primary antibody incubation

  • Optimize power and duration for S. pombe cells specifically

  • Compare results with conventional room temperature or 4°C incubations

How can researchers troubleshoot non-specific binding when using SPAC17G8.11c antibodies?

When encountering non-specific binding with SPAC17G8.11c antibodies:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time or concentration

  • Add 0.1-0.5% detergent (Tween-20, Triton X-100) to reduce hydrophobic interactions

• Antibody dilution series:

  • Perform a wide-range titration (1:100 to 1:10,000)

  • Correlate antibody concentration with signal-to-noise ratio

  • Determine minimum concentration that maintains specific signal

• Preabsorption protocols:

  • Preincubate antibody with recombinant protein or lysates from knockout strains

  • Use peptide competition assays with immunizing peptide

  • Employ subtractive approaches comparing wildtype and knockout samples

• Wash optimization:

  • Increase stringency with higher salt concentration (150-500 mM NaCl)

  • Add low concentrations of SDS (0.01-0.1%) to washes

  • Extend wash durations and increase wash frequency

What quality control metrics should be applied to ensure SPAC17G8.11c antibody consistency between experiments?

To ensure experimental consistency with SPAC17G8.11c antibodies:

• Standard curve generation:

  • Create standard curves using recombinant SPAC17G8.11c protein

  • Document antibody lot-to-lot variation through comparative analysis

  • Establish internal reference standards for normalization

• Reproducibility assessment:

  • Implement technical replicates (minimum n=3) for all critical experiments

  • Calculate coefficients of variation (CV) for quantitative assays (<15% typically acceptable)

  • Maintain detailed laboratory records of performance across experiments

• Regular antibody validation:

  • Revalidate antibodies after extended storage periods

  • Test against positive and negative controls with each new experiment

  • Compare results across different antibody lots when available

• Documentation standards:

  • Record batch numbers, storage conditions, and thaw cycles

  • Maintain control sample results across experimental series

  • Implement standardized protocols with explicit quality control checkpoints

How should researchers interpret contradictory results when using different detection methods with SPAC17G8.11c antibodies?

When facing contradictory results across detection methods:

• Systematic comparison framework:

  • Create a structured analysis comparing results from each method

  • Document detailed experimental conditions for each approach

  • Evaluate detection sensitivity limits for each method

• Epitope accessibility considerations:

  • Different preparation methods may affect epitope exposure differently

  • Consider protein conformation differences between native and denatured states

  • Assess whether post-translational modifications might affect antibody recognition

• Orthogonal validation approaches:

  • Implement non-antibody-based methods (e.g., mass spectrometry)

  • Use genetic approaches (knockouts, tagged proteins) to validate antibody results

  • Apply RNA expression analysis (RT-PCR, RNA-Seq) to correlate with protein detection

• Resolution strategies:

  • For functional studies, prioritize results from assays closest to native conditions

  • When quantifying, consider using multiple antibodies targeting different epitopes

  • Reconcile differences by identifying method-specific limitations or artifacts

How can SPAC17G8.11c antibodies be integrated into multi-omics approaches for comprehensive functional characterization?

For integrating SPAC17G8.11c antibodies into multi-omics approaches:

• Immunoprecipitation coupled with RNA-Seq (RIP-Seq):

  • Use SPAC17G8.11c antibodies to precipitate protein-RNA complexes

  • Sequence associated RNAs to identify potential regulatory interactions

  • Correlate with transcriptome-wide RNA expression data

• ChIP-Seq applications:

  • If SPAC17G8.11c has potential DNA-binding properties, perform chromatin immunoprecipitation

  • Map genome-wide binding sites and correlate with gene expression patterns

  • Integrate with epigenomic data to identify regulatory patterns

• Proteomics integration:

  • Compare immunoprecipitation-mass spectrometry data with global proteome changes

  • Identify condition-specific interaction networks

  • Correlate protein abundance with post-translational modification states

• Spatial and temporal dynamics:

  • Use antibodies for time-course immunofluorescence during cell cycle or stress responses

  • Correlate localization changes with functional outcomes

  • Integrate with live-cell imaging using fluorescent protein fusions

What considerations are important when using SPAC17G8.11c antibodies for quantitative comparative proteomics?

For quantitative comparative proteomics with SPAC17G8.11c antibodies:

• Standardization approaches:

  • Develop absolute quantification methods using isotope-labeled peptide standards

  • Implement normalization strategies for cross-sample comparisons

  • Establish dynamic range limitations for quantitative measurements

• Experimental design optimization:

  • Include biological replicates (minimum n=3) for statistical power

  • Design factorial experiments to assess multiple variables simultaneously

  • Implement randomization and blinding where applicable

• Data analysis considerations:

  • Apply appropriate statistical methods for comparative analysis

  • Utilize power analysis to determine sample size requirements

  • Account for technical variation in statistical models

• Validation strategies:

  • Confirm key findings with orthogonal quantitative methods

  • Correlate protein abundance with functional readouts

  • Implement targeted proteomics approaches (PRM/MRM) for selected targets

How can researchers develop custom modifications to SPAC17G8.11c antibodies for specialized applications?

For developing customized SPAC17G8.11c antibody modifications:

• Antibody fragmentation approaches:

  • Generate Fab fragments for improved tissue penetration

  • Produce F(ab')₂ fragments to eliminate Fc-mediated interactions

  • Optimize digestion conditions specifically for anti-SPAC17G8.11c antibodies

• Site-specific conjugation strategies:

  • Implement enzymatic approaches (sortase, transglutaminase) for controlled attachment

  • Utilize click chemistry for bioorthogonal modifications

  • Compare random versus site-specific labeling effects on binding properties

• Novel detection modalities:

  • Develop proximity-based split enzyme systems for protein interaction studies

  • Create photoactivatable antibody derivatives for spatiotemporal control

  • Design antibody-DNA conjugates for super-resolution microscopy applications

• Validation requirements:

  • Establish comprehensive binding kinetics before and after modifications

  • Compare specificity profiles of modified versus unmodified antibodies

  • Validate functional outcomes in biological assays relevant to S. pombe research

How can SPAC17G8.11c antibodies contribute to evolutionary studies across fungal species?

SPAC17G8.11c antibodies can facilitate evolutionary studies across fungi through:

• Comparative expression analysis:

  • Detect orthologous proteins across related fungal species

  • Quantify expression level differences in conserved pathways

  • Correlate protein conservation with functional conservation

• Structural conservation assessment:

  • Use epitope recognition patterns to infer structural conservation

  • Compare subcellular localization across species using immunofluorescence

  • Analyze post-translational modification conservation using modification-specific antibodies

• Experimental approaches:

  • Perform parallel immunoprecipitations across multiple fungal species

  • Use antibodies to purify protein complexes for comparative interactome studies

  • Implement cross-species complementation studies with antibody-based validation

• Phylogenetic applications:

  • Map antibody cross-reactivity onto phylogenetic trees

  • Correlate epitope conservation with evolutionary distance

  • Use antibody recognition patterns to supplement sequence-based phylogenies

What methodological adaptations are needed when using SPAC17G8.11c antibodies in different fungal species?

When adapting SPAC17G8.11c antibody protocols to different fungal species:

• Cell wall considerations:

  • Optimize enzymatic digestion for different cell wall compositions

  • Adjust incubation times based on cell wall thickness in target species

  • Consider species-specific cell wall inhibitors during sample preparation

• Extraction buffer modifications:

  • Adjust buffer composition based on species-specific protein solubility

  • Optimize detergent concentrations for different membrane compositions

  • Adapt protease inhibitor cocktails to species-specific proteases

• Fixation protocol adjustments:

  • Test species-specific fixation times and concentrations

  • Compare cross-linking fixatives versus precipitating fixatives

  • Develop specialized penetration enhancement steps for thick-walled species

• Controls and validation:

  • Generate recombinant orthologous proteins as positive controls

  • Implement CRISPR/genetic knockout controls when available

  • Use heterologous expression systems for antibody validation

How might SPAC17G8.11c antibodies be applied in studying cellular stress responses in S. pombe?

Applications of SPAC17G8.11c antibodies in stress response research:

• Temporal expression profiling:

  • Track protein abundance changes during various stress conditions

  • Correlate with transcriptional data from stress response experiments

  • Develop quantitative assays for high-resolution time course studies

• Subcellular relocalization studies:

  • Monitor potential compartment-specific accumulation during stress

  • Combine with organelle markers for co-localization analysis

  • Implement live-cell compatible immunostaining approaches

• Post-translational modification analysis:

  • Develop modification-specific antibodies if relevant

  • Use IP-MS approaches to identify stress-induced modifications

  • Correlate modifications with functional changes during stress

• Protein-protein interaction dynamics:

  • Compare interactome changes between normal and stress conditions

  • Identify stress-specific binding partners

  • Develop FRET-based biosensors for real-time interaction monitoring

What are the emerging technologies that could enhance SPAC17G8.11c antibody applications in fungal research?

Emerging technologies enhancing antibody applications include:

• Microfluidic antibody applications:

  • Develop on-chip immunoassays for high-throughput phenotyping

  • Implement droplet-based single-cell antibody assays

  • Create gradient-based approaches for antibody optimization

• Advanced imaging modalities:

  • Apply expansion microscopy for improved spatial resolution

  • Implement light-sheet microscopy for whole-cell antibody mapping

  • Develop correlative light-electron microscopy protocols using immunogold labeling

• Computational antibody engineering:

  • Use machine learning to predict epitope accessibility

  • Develop computational tools for antibody cross-reactivity prediction

  • Create in silico models to optimize antibody-based purification strategies

• Single-molecule applications:

  • Implement antibody-based single-molecule tracking

  • Develop antibody-DNA origami conjugates for precise spatial positioning

  • Create antibody-based molecular tension sensors

How can SPAC17G8.11c antibodies contribute to understanding the function of previously uncharacterized genes in S. pombe?

SPAC17G8.11c antibodies can advance functional genomics through:

• Systematic localization studies:

  • Implement high-throughput immunofluorescence for uncharacterized gene products

  • Compare localization patterns with known functional protein classes

  • Create comprehensive subcellular location maps for functional inference

• Perturbation-based functional screening:

  • Monitor protein abundance changes during genetic or chemical perturbations

  • Correlate phenotypic outcomes with molecular changes

  • Identify functional relationships through co-regulated expression patterns

• Multi-layered data integration:

  • Combine antibody-based detection with transcriptomics and genetic interaction data

  • Develop integrated models for function prediction

  • Validate predictions through targeted genetic manipulation

• Conservation-based functional inference:

  • Compare localization and expression patterns with orthologs of known function

  • Use antibodies to validate predicted functional domains

  • Implement cross-species complementation studies with antibody validation