SPAC17H9.06c Antibody

What is the SPAC17H9.06c protein and why is it significant in research?

SPAC17H9.06c is a conserved fungal protein found in Schizosaccharomyces pombe (fission yeast) . Its significance in research stems from its conservation across fungal species, suggesting an important biological role that has been maintained throughout evolution. The protein is encoded by the gene with Entrez Gene ID 2542297 and UniProt Number O13803 . Studying this protein can provide insights into fundamental cellular processes in fungi, particularly in S. pombe, which serves as an important model organism for understanding eukaryotic cell biology and genetics. The availability of specific antibodies against this protein enables researchers to investigate its expression, localization, and function in various experimental contexts.

What are the key specifications and validations necessary for the SPAC17H9.06c Antibody?

The SPAC17H9.06c Antibody (CSB-PA521044XA01SXV-2) is a rabbit polyclonal antibody raised against a recombinant Schizosaccharomyces pombe (strain 972 / ATCC 24843) SPAC17H9.06c protein . For proper validation, researchers should verify:

• Antibody specificity: Confirm target recognition using the provided 200μg antigen as a positive control and the 1ml pre-immune serum as a negative control .

• Application validation: The antibody is validated for ELISA and Western Blot (WB) applications .

• Cross-reactivity assessment: The antibody is specifically reactive to yeast species .

• Purification method: The antibody has undergone Antigen Affinity purification, which enhances specificity .

• Storage conditions: Maintain at -20°C or -80°C for optimal stability and performance .

When designing experiments, researchers should include proper controls and validation steps to ensure that any observed signals truly represent the SPAC17H9.06c protein rather than non-specific binding.

How should experimental controls be designed when using SPAC17H9.06c Antibody?

When designing experiments with the SPAC17H9.06c Antibody, a systematic approach to controls is essential:

• Positive controls: Utilize the provided 200μg antigen that comes with the antibody kit as a positive control to confirm antibody functionality . This ensures that the absence of signal in experimental samples is not due to antibody failure.

• Negative controls: Implement the supplied 1ml pre-immune serum as a negative control to establish baseline and non-specific binding levels . This helps distinguish true signals from background noise.

• Technical controls: Include loading controls (such as housekeeping proteins) in Western blot applications to normalize protein amounts across samples.

• Biological controls: Compare wild-type strains with SPAC17H9.06c knockout strains (if available) to validate antibody specificity in biological contexts.

• Treatment controls: For experimental manipulations, include untreated samples to establish baseline expression levels.

This systematic control design follows established principles for experimental design where manipulating the independent variable (e.g., experimental conditions) allows for proper assessment of effects on the dependent variable (e.g., SPAC17H9.06c protein levels) .

What is the optimal Western blot protocol for detecting SPAC17H9.06c protein?

For optimal Western blot detection of SPAC17H9.06c protein using the polyclonal antibody, follow this methodological approach:

• Sample preparation:

  • Extract proteins from S. pombe using spheroblasting technique to efficiently disrupt the cell wall

  • Include protease inhibitors to prevent protein degradation

  • Determine protein concentration using a reliable method (e.g., Bradford assay)

• Gel electrophoresis:

  • Use an appropriate percentage SDS-PAGE gel based on the molecular weight of SPAC17H9.06c

  • Load 20-50μg of total protein per lane

  • Include molecular weight markers and positive controls (provided antigen)

• Transfer and blocking:

  • Transfer proteins to PVDF or nitrocellulose membrane

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

• Antibody incubation:

  • Dilute primary SPAC17H9.06c antibody (determine optimal dilution through titration, typically starting at 1:1000)

  • Incubate overnight at 4°C with gentle agitation

  • Wash membrane 3-5 times with TBST

  • Incubate with appropriate HRP-conjugated secondary anti-rabbit antibody

• Detection and analysis:

  • Develop using ECL substrate

  • Image using a digital imaging system

  • Quantify band intensity using appropriate software, normalizing to loading controls

This protocol incorporates standard Western blot methodologies while accounting for the specific properties of the SPAC17H9.06c antibody and S. pombe samples.

How can SPAC17H9.06c Antibody be used in protein localization studies?

For sophisticated protein localization studies of SPAC17H9.06c in S. pombe, researchers can implement the following methodological framework:

• Immunofluorescence microscopy:

  • Fix S. pombe cells with 3.7% formaldehyde

  • Permeabilize cell wall using zymolyase or lysing enzymes

  • Block with BSA to prevent non-specific binding

  • Incubate with primary SPAC17H9.06c antibody (1:100-1:500 dilution)

  • Apply fluorophore-conjugated secondary antibody

  • Counterstain with DAPI for nuclear visualization

  • Image using confocal microscopy for high-resolution localization

• Subcellular fractionation validation:

  • Implement proteinase K protection assays to determine membrane association

  • Perform Western blot analysis on different cellular fractions

  • Compare SPAC17H9.06c distribution against known compartment markers

• Co-localization studies:

  • Combine SPAC17H9.06c antibody staining with markers for specific organelles

  • Calculate co-localization coefficients (Pearson's or Mander's)

  • Conduct proximity ligation assays (PLA) to detect protein-protein interactions

• Live cell imaging complementation:

  • Correlate antibody-based localization with data from GFP-tagged SPAC17H9.06c studies

  • Validate findings across different growth conditions and cell cycle stages

This comprehensive approach provides multiple lines of evidence for protein localization, minimizing artifacts from any single method.

What strategies can address potential cross-reactivity issues with SPAC17H9.06c Antibody?

Addressing cross-reactivity concerns requires systematic validation and troubleshooting approaches:

• Pre-absorption validation:

  • Incubate the antibody with excess antigen (provided 200μg)

  • Apply to identical samples in parallel experiments

  • Compare signal patterns between absorbed and non-absorbed antibody

  • Diminished signals after pre-absorption indicate specific binding

• Knockout/knockdown controls:

  • Generate SPAC17H9.06c knockout strains using CRISPR-Cas9 or traditional methods

  • Note that if SPAC17H9.06c is essential for cell viability, conditional systems may be required

  • Compare antibody signals between wild-type and knockout samples

  • True-positive signals should disappear in knockout samples

• Epitope mapping:

  • Determine the specific peptide sequence recognized by the antibody

  • Perform BLAST analysis to identify proteins with similar epitopes

  • Test antibody against recombinant proteins with similar sequences

• Cross-species validation:

  • Test antibody reactivity against protein extracts from related yeast species

  • Compare signal patterns to predicted sequence conservation

• Alternative antibody comparison:

  • If available, compare results with antibodies targeting different epitopes of SPAC17H9.06c

  • Consistent results across different antibodies increase confidence in specificity

These approaches collectively provide strong evidence for antibody specificity, enabling confident interpretation of experimental results.

How can SPAC17H9.06c Antibody be used in chromatin immunoprecipitation (ChIP) experiments?

While the SPAC17H9.06c antibody is primarily validated for ELISA and Western blot applications , its adaptation for ChIP requires careful optimization and validation:

• ChIP protocol optimization:

  • Cross-link S. pombe cells with 1% formaldehyde for 10-15 minutes

  • Lyse cells and sonicate chromatin to 200-500bp fragments

  • Pre-clear chromatin with protein A/G beads

  • Immunoprecipitate with 5-10μg SPAC17H9.06c antibody

  • Include IgG control immunoprecipitation

  • Purify DNA and analyze by qPCR or sequencing

• Antibody validation for ChIP:

  • Perform Western blot on input samples to confirm protein detection

  • Include known non-target regions as negative controls in qPCR analysis

  • If SPAC17H9.06c function suggests DNA binding, target suspected binding regions

  • Compare enrichment to published ChIP-seq datasets if available

• Optimization considerations:

  • Titrate antibody amounts (2-10μg per reaction)

  • Test different cross-linking conditions

  • Optimize sonication parameters for efficient chromatin shearing

  • Consider dual cross-linking with disuccinimidyl glutarate (DSG) for improved protein-protein fixation

• Data analysis approach:

  • Calculate percent input or fold enrichment relative to IgG control

  • Identify statistically significant peaks using appropriate algorithms

  • Perform motif analysis on enriched regions

  • Integrate with transcriptomic data to establish functional relevance

This methodological framework provides a starting point for adapting the SPAC17H9.06c antibody for ChIP applications, although further optimization may be required.

What are the considerations for using SPAC17H9.06c Antibody in mass spectrometry-based interactome studies?

For interactome studies utilizing mass spectrometry with SPAC17H9.06c antibody, consider this comprehensive approach:

• Immunoprecipitation optimization:

  • Utilize affinity-purified SPAC17H9.06c antibody for higher specificity

  • Cross-link antibody to beads to prevent antibody contamination in samples

  • Include pre-immune serum control IPs to establish background binding

  • Implement stringent washing protocols to minimize non-specific binding

• Sample preparation for MS:

  • Elute immunoprecipitated complexes with appropriate buffers

  • Process samples following standardized protocols for digestion with trypsin

  • Prepare samples following specific MS guidelines to minimize contaminants

• Experimental design considerations:

  • Implement label-free or isotope labeling approaches (SILAC, TMT, iTRAQ)

  • Include multiple biological replicates (minimum 3)

  • Incorporate quantitative controls to distinguish true interactors from background

  • Consider native conditions versus cross-linked samples for different interaction types

• Data analysis strategy:

  • Filter against common contaminant databases

  • Apply statistical methods to identify significantly enriched proteins

  • Implement network analysis to visualize and interpret protein-protein interactions

  • Validate key interactions through orthogonal methods (co-IP, PLA, Y2H)

• Validation of interactions:

  • Confirm novel interactions using reciprocal IPs

  • Perform domain mapping to identify interaction regions

  • Assess functional relevance through genetic or biochemical assays

This methodology integrates principles from both immunoprecipitation and mass spectrometry to reliably identify the SPAC17H9.06c interactome.

How can experimental design be optimized when studying SPAC17H9.06c in different genetic backgrounds?

When examining SPAC17H9.06c across different genetic backgrounds, a systematic experimental design is essential:

• Strain selection and validation:

  • Choose appropriate S. pombe genetic backgrounds (e.g., wild-type 972/ATCC 24843)

  • Include strains with relevant genetic modifications (deletions, mutations)

  • Verify strain genotypes through PCR or sequencing

  • Consider using different strain backgrounds, similar to approaches used in other studies

• Control implementation:

  • Establish baseline SPAC17H9.06c expression in wild-type strains

  • Include isogenic controls for each genetic background

  • Maintain consistent growth conditions across all strains

  • Consider using strains with tagged SPAC17H9.06c as additional controls

• Experimental variables management:

  • Control for extraneous variables that might influence results :

    • Growth phase and cell density

    • Media composition and temperature

    • Time of sample collection

    • Extraction method consistency

• Data collection approach:

  • Implement a randomized or blocked experimental design

  • Include sufficient biological and technical replicates (minimum 3)

  • Blind researchers to sample identity during analysis when possible

  • Collect data systematically across all conditions

• Statistical analysis framework:

  • Apply appropriate statistical tests based on data distribution

  • Control for multiple testing when examining many genetic backgrounds

  • Consider interaction effects between genetic background and treatments

  • Report effect sizes in addition to statistical significance

This methodological framework ensures robust, reproducible results when investigating SPAC17H9.06c across different genetic contexts.

What are the best practices for quantifying SPAC17H9.06c expression levels in response to environmental stressors?

For rigorous quantification of SPAC17H9.06c expression under environmental stress conditions:

• Stress condition standardization:

  • Define precise stress parameters (duration, intensity, application method)

  • Include gradients of stress intensity where appropriate

  • Apply treatments at consistent cell density and growth phase

  • Include recovery time points to assess temporal dynamics

• Protein quantification methodology:

  • Implement Western blotting with the SPAC17H9.06c antibody for protein levels

  • Include loading controls resistant to the applied stressors

  • Apply densitometric analysis with appropriate normalization

  • Consider supplementing with quantitative techniques like ELISA

• Transcriptional analysis integration:

  • Perform RT-qPCR for SPAC17H9.06c mRNA quantification

  • Compare protein and mRNA dynamics to identify post-transcriptional regulation

  • Reference established microarray hybridization protocols for broader analysis

• Experimental design approach:

  • Implement factorial designs when examining multiple stressors

  • Include time-course sampling for dynamic responses

  • Ensure sufficient biological replicates (minimum 3-5)

  • Include positive controls (stressors with known effects on other proteins)

• Data analysis framework:

  • Apply appropriate statistical models for time-course data

  • Consider non-linear responses to stress intensity

  • Test for interaction effects between different stressors

  • Correlate SPAC17H9.06c responses with physiological or cellular outcomes

This comprehensive approach enables reliable quantification of SPAC17H9.06c expression changes while controlling for confounding factors in stress response experiments.

How can researchers troubleshoot weak or inconsistent signals when using SPAC17H9.06c Antibody?

When facing weak or inconsistent signals, implement this systematic troubleshooting approach:

• Antibody optimization:

  • Titrate antibody concentration (typically 1:500 to 1:5000 for Western blots)

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

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Verify antibody viability through storage history and freeze-thaw cycles

• Sample preparation refinement:

  • Enhance protein extraction using optimized spheroblasting conditions

  • Incorporate additional protease inhibitors to prevent target degradation

  • Concentrate samples through precipitation or filtration if necessary

  • Verify protein integrity through Coomassie staining of replicate gels

• Technical parameter adjustment:

  • Modify transfer conditions (time, voltage, buffer composition)

  • Optimize detection system sensitivity (substrate exposure time, enhancers)

  • Adjust washing stringency to balance signal retention and background reduction

  • Consider alternative membranes (PVDF vs. nitrocellulose) based on protein properties

• Expression verification:

  • Confirm SPAC17H9.06c expression in your specific experimental conditions

  • Consider whether post-translational modifications might affect antibody recognition

  • Verify the presence of the protein using alternative detection methods if available

• Positive control implementation:

  • Use the provided 200μg antigen as a guaranteed positive control

  • Include samples with known high expression as reference points

  • Consider creating an overexpression system as a strong positive control

This methodical approach addresses the most common sources of signal problems when working with the SPAC17H9.06c antibody.

What special considerations apply when using SPAC17H9.06c Antibody in post-translational modification (PTM) studies?

When investigating post-translational modifications of SPAC17H9.06c, implement these specialized approaches:

• Epitope accessibility assessment:

  • Determine if the antibody epitope overlaps with potential PTM sites

  • Test whether PTMs might interfere with antibody recognition

  • Consider using denaturing conditions to expose buried epitopes

• Sample preparation refinement:

  • Include appropriate phosphatase or deubiquitinase inhibitors based on target PTMs

  • Implement PTM-preserving extraction protocols

  • Consider subcellular fractionation to enrich for modified forms

  • Apply PTM enrichment strategies (e.g., phosphopeptide enrichment)

• Detection strategy optimization:

  • Combine SPAC17H9.06c antibody with PTM-specific antibodies in sequential probing

  • Implement Phos-tag gels for phosphorylation studies

  • Use mobility shift assays to detect PTM-induced changes

  • Consider 2D gel electrophoresis to separate modified forms

• Mass spectrometry integration:

  • Immunoprecipitate SPAC17H9.06c using the antibody

  • Process samples using PTM-preserving protocols

  • Analyze using LC-MS/MS with appropriate fragmentation methods

  • Apply software specifically designed for PTM identification and localization

• Validation approach:

  • Generate site-specific mutants to confirm PTM sites

  • Use inhibitors or activators of relevant modifying enzymes

  • Compare PTM profiles across different conditions

  • Correlate PTMs with functional outcomes

This methodological framework enables comprehensive analysis of SPAC17H9.06c post-translational modifications while addressing the technical challenges inherent in PTM research.

How can SPAC17H9.06c Antibody be incorporated into high-throughput screening approaches?

For integrating SPAC17H9.06c antibody into high-throughput screening platforms:

• Assay miniaturization and adaptation:

  • Optimize antibody concentrations for microplate formats

  • Develop ELISA protocols with minimal sample requirements

  • Adapt Western blot protocols to dot blot formats for higher throughput

  • Consider automated liquid handling for consistent sample processing

• Screening platform selection:

  • Evaluate cell-based assays using immunofluorescence with automated imaging

  • Develop protein array applications for interaction screening

  • Implement bead-based assays for multiplexed detection

  • Consider label-free detection systems for real-time analysis

• Quality control implementation:

  • Include positive and negative controls on each plate/array

  • Incorporate internal standards for normalization

  • Calculate Z' factor to assess assay quality

  • Develop automated data analysis pipelines

• Validation strategy:

  • Confirm hits using orthogonal methods

  • Implement dose-response analysis for positive compounds

  • Develop secondary assays to eliminate false positives

  • Correlate screening results with functional outcomes

• Data management approach:

  • Implement structured data storage systems

  • Develop visualization tools for complex datasets

  • Apply appropriate statistical methods for hit identification

  • Integrate results with existing databases and knowledge

This comprehensive framework enables efficient integration of SPAC17H9.06c antibody into various high-throughput screening platforms while maintaining data quality and reliability.