SPAC22A12.14c Antibody

What is SPAC22A12.14c protein and what is its significance in fission yeast research?

SPAC22A12.14c is a BSD domain-containing protein found in Schizosaccharomyces pombe (strain 972 / ATCC 24843), commonly known as fission yeast. The protein is identified in the UniProt database with accession number O13905 . As a BSD domain-containing protein, it likely plays a role in transcriptional regulation or chromatin interactions.

While its exact function remains under investigation, studying this protein contributes to our understanding of fundamental cellular processes that are evolutionarily conserved between yeast and humans. Given that proteins controlling core cellular functions are evolutionarily conserved, research on SPAC22A12.14c can provide insights into "deep homology" that exists across species .

What are the recommended experimental applications for SPAC22A12.14c antibody?

Based on available literature and product information, SPAC22A12.14c antibody is primarily used in the following applications:

• Western blotting (WB): For detecting the native protein in cell lysates

• Enzyme-linked immunosorbent assay (ELISA): For quantitative detection of the protein

• Immunoprecipitation (IP): For isolating protein complexes containing SPAC22A12.14c

The antibody has been tested and validated for these applications specifically with fission yeast samples . When designing experiments, researchers should note that this polyclonal antibody was raised in rabbits using recombinant Schizosaccharomyces pombe SPAC22A12.14c protein as the immunogen .

How should SPAC22A12.14c antibody be stored to maintain optimal activity?

For proper maintenance of antibody activity:

• Short-term storage (up to 2 weeks): Maintain refrigerated at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

The antibody is typically supplied in a liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . This formulation helps maintain stability during storage periods.

What controls should be included when validating SPAC22A12.14c antibody specificity?

When validating antibody specificity, include the following controls:

• Positive control: Wild-type S. pombe lysate expressing SPAC22A12.14c

• Negative control: Either:

  • Lysate from a SPAC22A12.14c deletion strain (if available)

  • Preincubation of the antibody with excess recombinant SPAC22A12.14c protein to block specific binding

• Cross-reactivity control: Lysates from related yeast species to assess potential cross-reactivity

• Secondary antibody control: Omit primary antibody to check for non-specific binding of secondary antibody

For gene deletion verification, techniques similar to those used in yeast network analysis studies can be employed .

What is the recommended protocol for Western blotting using SPAC22A12.14c antibody?

The following protocol is recommended for Western blotting:

• Sample preparation:

  • Harvest S. pombe cells in log phase

  • Lyse cells using glass bead disruption in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% NP-40) with protease inhibitors

  • Clear lysate by centrifugation (13,000 rpm, 15 min, 4°C)

• Gel electrophoresis and transfer:

  • Separate 20-40 μg of total protein by SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane

• Antibody incubation:

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

  • Incubate with SPAC22A12.14c antibody at 1:1000 dilution overnight at 4°C

  • Wash 3× with TBST

  • Incubate with HRP-conjugated secondary antibody (anti-rabbit) at 1:5000 for 1 hour

  • Wash 3× with TBST

• Detection:

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expected molecular weight should be confirmed based on the protein's amino acid sequence

This protocol is adapted from standard methods used for yeast protein detection in published research .

How can SPAC22A12.14c antibody be used effectively in immunoprecipitation studies?

For effective immunoprecipitation:

• Pre-clearing step:

  • Incubate 1 mg of cell lysate with 20 μl of Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 2-5 μg of SPAC22A12.14c antibody to pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add 30 μl of fresh Protein A/G beads and incubate for 3 hours at 4°C

  • Collect beads by centrifugation and wash 4× with lysis buffer

  • Elute bound proteins by boiling in SDS sample buffer

• Analysis options:

  • Western blotting to confirm specific precipitation

  • Mass spectrometry to identify interaction partners

When identifying novel protein interactions, consider analytical approaches similar to those used in identifying components of protein complexes in fission yeast, as demonstrated in previous research .

How can SPAC22A12.14c antibody be integrated into YANA (Yeast Augmented Network Analysis) studies?

YANA is a systems approach that leverages fission yeast to identify human disease gene networks. To incorporate SPAC22A12.14c antibody in such studies:

• Network identification:

  • Use SPAC22A12.14c antibody in immunoprecipitation coupled with mass spectrometry to identify protein interaction networks

  • Compare these networks with human homolog networks using bioinformatics approaches

• Functional validation:

  • After identifying potential interacting partners, use genetic methods (deletion, overexpression) to validate functional relationships

  • Construct genetic interaction maps as demonstrated in previous YANA studies

• Data integration:

  • Integrate your findings with existing protein-protein interaction databases

  • Use computational approaches to predict potential human disease gene networks

The power of this approach lies in combining synthetic genetics in a simple model system to identify disease networks that can potentially be targeted therapeutically in humans .

What methodological approaches can resolve inconsistent results when using SPAC22A12.14c antibody?

When facing inconsistent results:

• Antibody validation:

  • Perform epitope mapping to confirm the antibody binds to the expected region

  • Consider using alternative antibody lots or sources

  • Validate specificity using knockout or knockdown controls

• Sample preparation optimization:

  • Evaluate different lysis methods (chemical vs. mechanical disruption)

  • Test various buffer compositions to preserve protein integrity

  • Consider native vs. denaturing conditions based on the experimental goal

• Experimental parameters:

  • Systematically adjust antibody concentration, incubation time, and temperature

  • Test different blocking agents to reduce background

  • Optimize washing conditions to maintain specific binding while reducing non-specific interactions

• Data analysis:

  • Use quantitative methods to compare results across experiments

  • Apply statistical analysis to determine significance of observed differences

  • Document all experimental conditions meticulously to identify variables affecting outcomes

These approaches are based on methodologies used in resolving antibody-related challenges in research settings .

How does post-translational modification of SPAC22A12.14c affect antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition:

• Phosphorylation effects:

  • Recent phosphoproteomic studies have revealed extensive fluctuations in global phosphorylation in response to nutrient stress in fission yeast

  • If SPAC22A12.14c contains phosphorylation sites, antibody recognition may be affected depending on the cellular state

  • Consider using phosphatase treatment of samples to determine if phosphorylation affects antibody binding

• Other potential PTMs:

  • Ubiquitination, sumoylation, or acetylation may alter epitope accessibility

  • If these modifications occur at or near the epitope recognized by the antibody, detection efficiency may vary

• Experimental approaches:

  • Compare antibody recognition in different growth conditions known to alter PTM status

  • Use PTM-specific inhibitors to determine their impact on antibody binding

  • Consider generating modification-specific antibodies if a particular PTM is confirmed

Understanding the relationship between PTMs and antibody recognition is crucial for accurate interpretation of experimental results, particularly in stress response studies .

How can SPAC22A12.14c antibody be used to study transcriptional regulation in fission yeast?

To investigate transcriptional regulation:

• Chromatin immunoprecipitation (ChIP):

  • Use SPAC22A12.14c antibody for ChIP to identify DNA binding sites

  • Protocol adaptation:

    • Crosslink cells with 1% formaldehyde for 15 minutes

    • Lyse cells and sonicate to fragment chromatin

    • Immunoprecipitate with SPAC22A12.14c antibody

    • Purify DNA and analyze by qPCR or sequencing

• Transcription factor complex analysis:

  • Combine immunoprecipitation with mass spectrometry to identify factors associating with SPAC22A12.14c

  • Research has shown that BSD domain-containing proteins may interact with transcriptional machinery

• Integration with RNA Pol II studies:

  • Correlate SPAC22A12.14c binding with RNA Pol II occupancy data

  • Previous studies have analyzed RNA Pol II occupancy in relation to translation efficiency in fission yeast

This approach builds on methods used to study other factors involved in transcriptional regulation in S. pombe .

What are the considerations when comparing SPAC22A12.14c antibody results across different fission yeast strains?

When comparing results across strains:

• Strain-specific variations:

  • Wild-type strains may retain more natural S. pombe phenotypes compared to lab strains

  • Consider genetic background effects on protein expression and modification

• Growth condition standardization:

  • Maintain identical growth conditions (media, temperature, growth phase)

  • Document any strain-specific growth characteristics

• Data normalization approaches:

  • Use appropriate housekeeping proteins as loading controls

  • Consider quantitative proteomics approaches for more accurate comparisons

• Phenotypic correlation:

  • Connect antibody-based observations with phenotypic differences between strains

  • Consider whether strain-specific phenotypes like flocculation might affect protein expression or localization

A systematic approach to these considerations will help ensure valid comparisons and identify genuine strain-dependent differences in SPAC22A12.14c expression or function.

How can datasets generated using SPAC22A12.14c antibody be integrated with other -omics data?

For effective data integration:

• Multi-omics data collection:

  • Generate parallel datasets such as:

    • Proteomics data from immunoprecipitation studies

    • Transcriptomic data (RNA-seq) to correlate with protein abundance

    • Functional genomics data from genetic screens

• Bioinformatic approaches:

  • Apply network analysis to identify functional relationships

  • Use clustering algorithms to identify co-regulated genes/proteins

  • Implement machine learning approaches to predict function from integrated datasets

• Visualization methods:

  • Utilize tools like Cytoscape for network visualization

  • Create integrated heatmaps showing relationships across multiple datasets

  • Develop custom visualization approaches for specific scientific questions

This integration approach has been demonstrated in studies such as the TOR signaling phosphoproteome analysis in fission yeast, where researchers integrated multiple datasets to identify novel targets in cellular signaling pathways .

What are the most common artifacts when using SPAC22A12.14c antibody and how can they be distinguished from genuine signals?

Common artifacts and their resolution:

• Non-specific bands in Western blotting:

  • Cause: Cross-reactivity with proteins containing similar epitopes

  • Resolution: Use knockout controls, peptide competition assays, or alternative antibody clones

  • Distinction: Non-specific bands often remain present in knockout controls

• Background in immunofluorescence:

  • Cause: Inadequate blocking or secondary antibody cross-reactivity

  • Resolution: Optimize blocking conditions, increase washing steps, titrate antibody concentration

  • Distinction: Background staining typically lacks co-localization with expected cellular structures

• False positives in immunoprecipitation:

  • Cause: Proteins binding non-specifically to beads or antibody

  • Resolution: Include IgG control immunoprecipitations, use more stringent washing conditions

  • Distinction: Compare with mass spectrometry data from control samples

• Batch-to-batch variability:

  • Cause: Differences in antibody production

  • Resolution: Validate each new lot against previous successful experiments

  • Distinction: Systematic shifts in signal intensity across all samples with new antibody lot

These troubleshooting approaches are based on standard practices in antibody validation and quality control .

How can researchers determine optimal antibody concentration for different experimental applications?

Determination of optimal antibody concentration:

• Titration experiment design:

  Antibody Dilution	Western Blot	ELISA	Immunofluorescence	ChIP
  1:100	Test	Test	Test	Test
  1:500	Test	Test	Test	Test
  1:1000	Test	Test	Test	Test
  1:5000	Test	Test	Test	Test
  1:10000	Test	Test	Test	Test

• Evaluation criteria:

  • Signal-to-noise ratio: Calculate using image analysis software

  • Specific band intensity: Quantify relative to background

  • Reproducibility: Ensure consistent results across technical replicates

• Application-specific considerations:

  • Western blot: Lower concentrations often sufficient (1:1000-1:5000)

  • Immunofluorescence: May require higher concentrations (1:100-1:500)

  • ChIP: Often requires optimization with positive control regions

• Documentation:

  • Record all parameters including antibody lot, incubation time/temperature

  • Document manufacturer details as shown in reference materials for reproducibility

This methodical approach ensures optimal antibody usage while minimizing waste of valuable reagents.

What key information should be reported in publications using SPAC22A12.14c antibody to ensure reproducibility?

Essential reporting elements:

• Antibody specifications:

  • Complete source information (manufacturer, catalog number, lot number)

  • Antibody type (polyclonal/monoclonal), host species, and clonality

  • Storage conditions and any reconstitution details

• Validation methods:

  • Specific controls used to verify specificity

  • References to previous validation studies

  • Any additional validation performed by the authors

• Experimental protocols:

  • Detailed sample preparation procedures

  • Complete buffer compositions

  • Antibody dilutions, incubation times and temperatures

  • Detection methods with full parameters

• Data acquisition and analysis:

  • Image acquisition settings

  • Software used for quantification

  • Statistical methods applied

  • Raw data availability statement

Following these reporting guidelines aligns with best practices in antibody research and ensures that other researchers can accurately reproduce and build upon published findings .