SPAC13G6.09 Antibody

What is SPAC13G6.09 and why would researchers develop antibodies against it?

SPAC13G6.09 follows the systematic gene-naming convention for Schizosaccharomyces pombe (fission yeast), where SPAC indicates the species prefix for S. pombe, 13G6 represents the chromosomal locus (13th chromosome, G6 region), and 09 is the open reading frame (ORF) identifier. Researchers typically develop antibodies against S. pombe proteins to study their expression, localization, interactions, and functions within cellular processes. Antibodies enable visualization of proteins through techniques like immunofluorescence microscopy, western blotting, and immunoprecipitation, providing valuable insights into gene function in this model organism.

How should researchers validate a SPAC13G6.09 antibody before experimental use?

Validation of any S. pombe-specific antibody should follow a multi-step approach:

• Specificity testing: Perform western blot analysis comparing wild-type strains versus knockout or deletion mutants lacking SPAC13G6.09

• Cross-reactivity assessment: Test against homologous proteins using ELISA to ensure species specificity

• Functional validation: Correlate antibody binding with known gene knockout phenotypes

• Epitope mapping: Use peptide microarrays to identify precise binding regions

A comprehensive validation approach ensures reliable results in subsequent experiments and prevents misinterpretation of data due to non-specific binding.

What are the common applications for a SPAC13G6.09 antibody in yeast research?

These applications provide complementary data that collectively elucidate protein function within the broader cellular context of S. pombe.

How can researchers address potential cross-reactivity when using SPAC13G6.09 antibody in complex experimental systems?

Cross-reactivity represents a significant challenge when working with antibodies in complex systems. For SPAC13G6.09 antibodies:

• Pre-absorption controls: Incubate the antibody with purified recombinant SPAC13G6.09 protein prior to use in assays. Complete signal loss confirms specificity.

• Epitope competition assays: Include varying concentrations of the immunizing peptide during primary antibody incubation to demonstrate specific displacement.

• Orthogonal detection methods: Compare antibody-based detection with orthogonal techniques like mass spectrometry or genetic tagging methods.

• Cross-species validation: Test the antibody against related organisms with known sequence homology to identify potential cross-reactive epitopes.

For maximum confidence, researchers should implement multiple approaches and include appropriate negative controls (including isotype controls similar to those used for other antibody systems) .

What strategies exist for optimizing SPAC13G6.09 antibody performance in immunoprecipitation experiments?

Optimizing immunoprecipitation with yeast proteins requires careful consideration of several parameters:

• Lysis buffer optimization:

  • Test buffers with varying detergent concentrations (0.1-1% NP-40, Triton X-100)

  • Adjust salt concentrations (150-500 mM NaCl) to modulate stringency

  • Include appropriate protease and phosphatase inhibitors

• Antibody coupling approaches:

  • Direct coupling to protein A/G beads

  • Covalent coupling to reduce antibody contamination in mass spectrometry experiments

  • Pre-clearing lysates to reduce non-specific binding

• Incubation conditions:

  • Testing different temperatures (4°C vs. room temperature)

  • Varying incubation times (2 hours vs. overnight)

  • Implementing gentle agitation methods

• Wash stringency gradients:

  • Progressive increase in wash buffer stringency

  • Determination of minimum conditions that maintain specific interactions

The optimal protocol will vary depending on SPAC13G6.09's abundance, solubility properties, and interaction stability.

How should researchers interpret contradictory results between SPAC13G6.09 antibody-based detection and genetic approaches?

When antibody-based detection yields results that contradict genetic approaches:

• Validate antibody specificity: Re-confirm antibody specificity through additional controls, particularly gene deletion strains.

• Consider post-translational modifications: The antibody may recognize specific modified forms of the protein not represented in genetic models.

• Examine epitope accessibility: The three-dimensional conformation of the protein in different experimental contexts may affect epitope accessibility.

• Evaluate genetic compensation mechanisms: Genetic approaches may trigger compensatory mechanisms not present in antibody-based studies.

• Compare detection sensitivity thresholds: Antibody-based methods and genetic approaches have different detection limits that may explain apparent contradictions.

To resolve such contradictions, researchers should employ complementary approaches such as epitope tagging, mass spectrometry, or alternative antibodies targeting different epitopes of SPAC13G6.09.

What is the optimal protocol for using SPAC13G6.09 antibody in immunofluorescence of S. pombe cells?

The following protocol optimizes immunofluorescence detection of SPAC13G6.09 in fission yeast:

• Cell fixation:

  • Grow cells to mid-log phase (OD600 = 0.5-0.8)

  • Fix with 3.7% formaldehyde for 30 minutes at room temperature

  • Alternative fixation: 100% methanol at -20°C for 8 minutes for certain epitopes

• Cell wall digestion:

  • Treat with zymolyase (1mg/ml) in PEMS buffer for 30-60 minutes at 37°C

  • Monitor spheroplasting by phase-contrast microscopy

• Permeabilization:

  • 1% Triton X-100 in PBS for 5 minutes

  • Alternative: 0.1% SDS for 1 minute for certain nuclear proteins

• Blocking and antibody incubation:

  • Block with 5% BSA in PEMBAL buffer for 60 minutes

  • Incubate with primary antibody (optimal dilution determined empirically, typically 1:100-1:1000) overnight at 4°C

  • Wash 3x in PEMBAL

  • Incubate with fluorophore-conjugated secondary antibody (1:500) for 2 hours at room temperature

• Mounting and imaging:

  • Mount in anti-fade medium containing DAPI

  • Image using appropriate filter sets

For co-localization studies, compatible secondary antibodies (similar to the PE-conjugated format used for other immunostaining applications) should be selected to avoid spectral overlap .

How can researchers quantitatively assess SPAC13G6.09 protein levels in yeast extracts using western blotting?

For quantitative western blot analysis:

• Sample preparation:

  • Extract proteins using TCA precipitation or mechanical disruption with glass beads

  • Quantify total protein using Bradford or BCA assay

  • Normalize loading to 20-50μg total protein per lane

• Gel electrophoresis and transfer:

  • Select appropriate acrylamide percentage based on expected molecular weight

  • Use wet transfer system with methanol-containing buffer for optimal protein transfer

• Antibody incubation:

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

  • Incubate with primary antibody overnight at 4°C (1:1000 dilution recommended for initial testing)

  • Use HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Quantification approach:

  • Include serial dilutions of recombinant protein as standard curve

  • Use housekeeping proteins (e.g., actin, GAPDH) as loading controls

  • Employ fluorescent secondary antibodies for wider linear dynamic range when precise quantification is required

  • Analyze using software that corrects for background and normalizes to loading controls

• Statistical analysis:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA) based on experimental design

This approach provides both qualitative confirmation of specificity and accurate quantification of protein levels.

What experimental design would best determine if SPAC13G6.09 undergoes cell cycle-dependent modifications?

To investigate cell cycle-dependent modifications:

• Cell synchronization approaches:

  • Nitrogen starvation and release

  • Hydroxyurea block and release

  • Temperature-sensitive cdc mutants

  • Centrifugal elutriation for size-based separation

• Time-course sampling:

  • Collect samples at 15-20 minute intervals covering the full cell cycle (approximately 2-3 hours)

  • Process parallel samples for flow cytometry to confirm synchronization quality

  • Fix cells for microscopy to correlate with cell cycle phases

• Analytical methods:

  • Western blotting with mobility shift detection

  • Phospho-specific antibodies if phosphorylation is suspected

  • Immunoprecipitation followed by mass spectrometry to identify modifications

  • 2D gel electrophoresis to resolve modified isoforms

• Controls and validation:

  • Include asynchronous culture controls

  • Use phosphatase treatment to confirm phosphorylation events

  • Compare with known cell cycle markers (e.g., Cdc13, Cdc2)

This comprehensive approach allows detection of transient modifications that might be diluted in asynchronous populations .

How should researchers address weak or absent signal when using SPAC13G6.09 antibody?

When facing weak or absent signals:

• Antibody-related factors:

  • Test multiple antibody concentrations (perform a titration series)

  • Extend primary antibody incubation time or temperature

  • Verify antibody storage conditions and expiration date

  • Consider different clones if available or epitope-specific antibodies targeting different regions

• Sample preparation optimization:

  • Modify extraction buffer composition (detergents, salt concentration)

  • Test alternative fixation methods for immunofluorescence

  • Enrich target protein through fractionation or immunoprecipitation

• Detection system enhancement:

  • Implement signal amplification methods (TSA, polymer-based detection)

  • Use more sensitive detection substrates (enhanced chemiluminescence)

  • Extend exposure times for western blotting

  • Optimize microscope settings for immunofluorescence

• Protein abundance and accessibility:

  • Confirm protein expression under current experimental conditions

  • Consider cellular compartment accessibility issues

  • Test epitope retrieval methods if applicable

These approaches systematically address the most common causes of signal problems when working with antibodies against low-abundance yeast proteins .

What comparative approach should researchers use when evaluating multiple SPAC13G6.09 antibody clones?

When evaluating multiple antibody clones:

This systematic evaluation framework allows researchers to select the optimal antibody clone for their specific experimental needs, rather than using a single metric for selection .

How can researchers determine if SPAC13G6.09 antibody is suitable for chromatin immunoprecipitation (ChIP) experiments?

To assess ChIP suitability:

• Preliminary evaluation:

  • Perform standard immunoprecipitation to confirm antibody binding under native conditions

  • Confirm antibody performance in formaldehyde-fixed samples by western blot

  • Assess antibody specificity via protein knockdown/knockout controls

• ChIP protocol optimization:

  • Test different crosslinking conditions (0.75-3% formaldehyde, 5-20 minutes)

  • Optimize sonication parameters for ideal chromatin fragment size (200-500bp)

  • Compare different antibody concentrations (2-10μg per reaction)

  • Evaluate various washing stringencies to minimize background

• ChIP-qPCR validation:

  • Design primers targeting regions with predicted binding (if known)

  • Include negative control regions (housekeeping genes)

  • Compare enrichment to IgG control

  • Calculate signal-to-noise ratios at target vs. control regions

• Quality metrics for success:

  • 4-fold enrichment over IgG control

  • <1% input recovery at negative regions

  • Reproducible enrichment patterns across replicates

  • Depletion of signal in genetic knockout controls

This approach ensures that the antibody can specifically recognize the SPAC13G6.09 protein in the context of chromatin and provides reliable data for downstream applications like ChIP-seq .

How can SPAC13G6.09 antibody be used in proximity labeling approaches to study protein-protein interactions?

SPAC13G6.09 antibody can be integrated with proximity labeling through the following approaches:

• BioID or TurboID fusion protein generation:

  • Create genetic fusions of SPAC13G6.09 with biotin ligase

  • Express in S. pombe under native or regulated promoters

  • Verify expression and functionality using the SPAC13G6.09 antibody

  • Identify interacting proteins via streptavidin pulldown and mass spectrometry

• Antibody-based proximity labeling:

  • Conjugate SPAC13G6.09 antibody with HRP or APEX2

  • Apply to fixed cells or cell extracts

  • Activate labeling with hydrogen peroxide and biotin-phenol

  • Identify labeled proteins by streptavidin pulldown and mass spectrometry

• Validation of proximity-based interactions:

  • Confirm selected interactions by co-immunoprecipitation with SPAC13G6.09 antibody

  • Perform reciprocal experiments using antibodies against identified partners

  • Correlate proximity labeling data with functional assays

• Spatiotemporal implementation:

  • Apply conditional expression systems to study interactions at specific cell cycle stages

  • Combine with subcellular fractionation to focus on specific compartments

This methodology enables the identification of both stable and transient protein interactions that might be missed by conventional co-immunoprecipitation approaches .

What considerations are important when using SPAC13G6.09 antibody for super-resolution microscopy?

When applying super-resolution microscopy techniques:

• Antibody characteristics for optimal imaging:

  • Validate high specificity to minimize background signal

  • Confirm sensitivity to detect low-abundance proteins

  • Select appropriate fluorophore-conjugated secondary antibodies with photostability properties suited to the super-resolution technique

• Sample preparation adaptations:

  • Optimize fixation protocols to preserve cellular ultrastructure

  • Test alternative permeabilization methods that maintain epitope accessibility

  • Consider cryosectioning for improved structural preservation

  • Test mounting media specifically formulated for super-resolution (containing oxygen scavengers for STORM/PALM)

• Technique-specific considerations:

  • For STORM/PALM: Select secondary antibodies with appropriate blinking characteristics

  • For SIM: Ensure high signal-to-noise ratio and consistent labeling

  • For STED: Choose fluorophores with suitable depletion properties

• Controls and validation:

  • Include knockout/knockdown controls to confirm specificity at super-resolution level

  • Perform correlative imaging with conventional techniques

  • Use known markers to validate colocalization claims

These considerations ensure that antibody-based detection is compatible with the higher resolution and technical demands of super-resolution microscopy techniques .

How might SPAC13G6.09 antibody be integrated with emerging single-cell proteomics approaches?

Integration with single-cell proteomics could involve:

• Antibody-based cell sorting:

  • Use SPAC13G6.09 antibody for cell isolation based on expression levels

  • Sort populations for downstream single-cell mass spectrometry

  • Correlate protein expression heterogeneity with cellular phenotypes

• Antibody-based microfluidic platforms:

  • Implement in microfluidic chips for single-cell protein detection

  • Combine with other antibodies for multiplexed analysis

  • Quantify protein levels across population distributions

• Mass cytometry applications:

  • Conjugate SPAC13G6.09 antibody with heavy metals for CyTOF analysis

  • Enable high-dimensional profiling in combination with other cellular markers

  • Assess correlations between SPAC13G6.09 expression and cellular states

• Spatial proteomics integration:

  • Use for highly multiplexed imaging approaches (CODEX, CycIF)

  • Correlate spatial distribution with functional organization

  • Implement in spatial transcriptomics-proteomics correlative studies

These emerging technologies would allow researchers to move beyond population averages and understand the role of SPAC13G6.09 in cellular heterogeneity and microenvironmental contexts .

What methodological advances might improve SPAC13G6.09 antibody sensitivity for detecting low-abundance forms of the protein?

Methodological advances for improved detection include:

• Signal amplification technologies:

  • Tyramide signal amplification (TSA) for immunohistochemistry and blotting

  • Proximity ligation assay (PLA) for detecting protein-protein interactions

  • Polymerized reporter enzyme mechanisms for enhanced sensitivity

  • Quantum dot conjugation for improved fluorescence detection

• Sample preparation enhancements:

  • Targeted protein enrichment using affinity purification

  • Subcellular fractionation to concentrate proteins from specific compartments

  • Optimized extraction methods for membrane or chromatin-associated proteins

  • Depletion of abundant proteins to enhance detection of low-abundance species

• Advanced detection systems:

  • Digital western blotting platforms with enhanced sensitivity

  • Single-molecule detection approaches

  • Nano-immunoassay methods for minimal sample requirements

  • Microfluidic antibody capture surfaces with real-time detection

These methodological advances could potentially lower detection thresholds by 10-100 fold compared to conventional techniques, enabling visualization and quantification of previously undetectable protein forms .