SPAC1A6.11 Antibody

What is SPAC1A6.11 and what cellular functions is it associated with?

SPAC1A6.11 is a protein in Schizosaccharomyces pombe (fission yeast) with the UniProt number Q9C114 and Entrez Gene ID 2542238 . While detailed functional characterization is still emerging in research literature, this protein is being studied primarily in S. pombe cellular processes. Methodologically, researchers investigating this protein typically employ a combination of genetic manipulation (gene knockouts, mutations) alongside immunological detection methods using antibodies like the polyclonal SPAC1A6.11 antibody to elucidate its localization, interaction partners, and functional role in yeast cellular pathways.

What applications are SPAC1A6.11 antibodies validated for?

The commercially available SPAC1A6.11 polyclonal antibody has been validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications . For researchers designing experiments, this means the antibody can reliably detect the target protein in denatured samples separated by gel electrophoresis (WB) and in solution-based immunoassays (ELISA). When implementing these methods, researchers should consider using the provided positive control antigens (200μg) to establish detection parameters and the pre-immune serum negative control to confirm specificity .

What is the recommended storage and handling protocol for SPAC1A6.11 antibody?

The SPAC1A6.11 antibody should be stored at either -20°C or -80°C to maintain its stability and binding efficacy . When handling the antibody, researchers should follow standard antibody handling protocols including:

• Minimizing freeze-thaw cycles (aliquot upon receipt)

• Centrifuging briefly before opening vials to collect all material

• Maintaining sterile technique when handling

• Diluting in appropriate buffers depending on the application

• Confirming activity periodically by running positive controls

How should I design a western blot experiment using SPAC1A6.11 antibody?

When designing a western blot experiment with SPAC1A6.11 antibody, researchers should consider the following methodological approach:

• Sample preparation: Extract proteins from S. pombe using appropriate lysis buffers (typically containing protease inhibitors)

• Protein quantification: Standardize protein concentration across samples

• Gel electrophoresis: Separate proteins using SDS-PAGE (typically 8-12% gels)

• Transfer: Transfer proteins to membrane (PVDF or nitrocellulose)

• Blocking: Block with 5% non-fat milk or BSA in TBST

• Primary antibody: Incubate with SPAC1A6.11 antibody at optimized dilution

• Secondary antibody: Use anti-rabbit IgG conjugated to HRP or fluorophore

• Detection: Visualize using chemiluminescence or fluorescence imaging

• Controls: Include the provided positive control antigen and pre-immune serum as a negative control

Optimization should include antibody dilution series and validation of specificity using known positive samples.

What are the best approaches for implementing ELISA with SPAC1A6.11 antibody?

For ELISA applications using SPAC1A6.11 antibody, consider the following protocol outline:

• Plate coating: Coat wells with purified target protein or cell lysate

• Blocking: Block with BSA or appropriate blocking buffer

• Primary antibody: Apply SPAC1A6.11 antibody in series of dilutions

• Secondary antibody: Use HRP-conjugated anti-rabbit antibody

• Detection: Develop using TMB or other appropriate substrate

• Quantification: Measure absorbance using spectrophotometer

• Controls: Include wells with pre-immune serum (negative control) and positive control antigen

For sandwich ELISA applications, researchers may need to pair this polyclonal antibody with a monoclonal antibody targeting a different epitope of the SPAC1A6.11 protein.

How can I optimize antibody concentration for different experimental applications?

Optimization of SPAC1A6.11 antibody concentration follows methodological principles similar to other polyclonal antibodies:

Researchers should perform preliminary experiments with serial dilutions of the antibody, comparing signal strength and specificity across conditions. The optimal concentration balances maximum specific signal with minimal background. Using the provided positive control antigens facilitates this optimization process .

How can SPAC1A6.11 antibody be used in combination with other technologies for protein interaction studies?

Advanced studies of protein interactions involving SPAC1A6.11 can employ multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): Use SPAC1A6.11 antibody to pull down the target protein complex from yeast lysates, followed by mass spectrometry or western blotting to identify interaction partners

• Proximity-based labeling: Employ BioID or APEX2 fused to SPAC1A6.11, followed by streptavidin pulldown and detection using SPAC1A6.11 antibody for validation

• FRET/BRET analysis: Use fluorescent or bioluminescent proteins fused to SPAC1A6.11 and potential interaction partners, with antibody validation of expression

• ChIP-seq: If SPAC1A6.11 has DNA-binding properties, chromatin immunoprecipitation using this antibody can map genomic binding sites

• Single-molecule imaging: Combine with fluorescent secondary antibodies for tracking protein dynamics in live or fixed cells

The affinity-purified nature of this polyclonal antibody makes it suitable for these applications, though each would require specific optimization protocols to minimize background and maximize specific detection .

What controls should be included when using SPAC1A6.11 antibody in comparative studies across yeast strains?

Robust comparative studies across yeast strains require comprehensive controls:

• Pre-immune serum control: The included pre-immune serum serves as a primary negative control to assess non-specific binding

• Genetic knockouts: SPAC1A6.11 deletion strains provide critical negative controls

• Protein expression controls: Housekeeping proteins should be measured to normalize loading across samples

• Species-specific controls: When examining cross-reactivity, include control samples from related species

• Epitope-blocked controls: Pre-incubation of antibody with purified target protein (the included antigen) to demonstrate specificity

• Sample preparation controls: Identically processed samples that differ only in the protein of interest

• Technical replicates: Multiple experimental runs to assess reproducibility

Proper implementation of these controls enables rigorous statistical analysis and increases confidence in observed differences between strains.

How can I assess potential cross-reactivity of SPAC1A6.11 antibody with homologous proteins?

Methodological approaches to assess cross-reactivity include:

• Sequence homology analysis: Computational comparison of the immunogen sequence (Recombinant S. pombe SPAC1A6.11) with homologous proteins in target and non-target species

• Western blot analysis: Testing the antibody against lysates from:

  • Wild-type S. pombe

  • SPAC1A6.11 knockout S. pombe

  • Related yeasts with homologous proteins

  • Comparing band patterns and intensities

• Competitive binding assays: Pre-incubating antibody with purified homologous proteins before the primary detection assay

• Epitope mapping: Determining the specific regions recognized by the polyclonal antibody to predict potential cross-reactivity

The antibody is specifically reactive with yeast species as indicated in the product information , but researchers should validate specificity for their particular experimental system, especially when working with closely related homologs.

What are common causes of weak or absent signal when using SPAC1A6.11 antibody in western blots?

When troubleshooting weak or absent signals with SPAC1A6.11 antibody, consider these methodological factors:

• Protein expression level: SPAC1A6.11 may be expressed at low levels in your specific conditions

  • Solution: Increase protein loading or use concentration techniques like immunoprecipitation before western blot

• Protein extraction efficiency: Yeast cell walls can hinder efficient protein extraction

  • Solution: Optimize lysis protocol with appropriate mechanical disruption (glass beads, sonication) and detergents

• Antibody concentration: Insufficient primary antibody

  • Solution: Titrate antibody using a dilution series; the affinity-purified nature of this antibody may require specific optimization

• Incubation conditions: Suboptimal binding conditions

  • Solution: Adjust temperature, time, and buffer composition for antibody incubation

• Detection sensitivity: Standard ECL may be insufficient

  • Solution: Use enhanced sensitivity substrates or switch to fluorescent detection systems

• Transfer efficiency: Poor transfer of protein to membrane

  • Solution: Verify transfer efficiency with reversible staining; optimize transfer conditions for proteins of similar size

Systematically testing these variables while including the positive control antigen will help identify the specific issue.

How can background signals be reduced when using SPAC1A6.11 antibody?

High background can compromise data quality. To reduce background when using this antibody:

• Optimize blocking conditions:

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

  • Increase blocking time or concentration

  • Include detergents like Tween-20 at appropriate concentrations

• Antibody dilution and incubation:

  • Increase antibody dilution systematically

  • Compare overnight incubation at 4°C vs. shorter incubations at room temperature

  • Add 0.1-0.5% BSA to antibody dilution buffer

• Washing optimization:

  • Increase number and duration of washes

  • Use higher detergent concentration in wash buffers

  • Consider different detergents (Tween-20, Triton X-100)

• Pre-adsorption technique:

  • Incubate antibody with non-target proteins or with a lysate from knockout strains

  • Use the pre-immune serum to identify non-specific binding patterns

• Secondary antibody optimization:

  • Test more highly cross-adsorbed secondary antibodies

  • Reduce secondary antibody concentration

The polyclonal nature of this antibody means it contains multiple antibody populations, which can sometimes contribute to background signals that require careful optimization.

What approaches can address inconsistent results between experimental replicates?

Inconsistency between replicates when using SPAC1A6.11 antibody may stem from several sources:

• Antibody storage and handling:

  • Aliquot antibody upon receipt to avoid repeated freeze-thaw cycles

  • Ensure proper storage at -20°C or -80°C as recommended

  • Check for contamination or degradation

• Sample preparation variability:

  • Standardize protein extraction methods

  • Implement rigorous protein quantification

  • Prepare fresh samples when possible

• Technical execution:

  • Develop standardized protocols with precise timing

  • Use automated systems where possible

  • Implement consistent transfer conditions

• Statistical considerations:

  • Determine appropriate sample size for statistical power

  • Apply appropriate statistical tests for your experimental design

  • Consider blocking experimental variables

• Environmental factors:

  • Control laboratory temperature and humidity

  • Standardize incubation equipment (shakers, rockers)

  • Use calibrated pipettes and validated reagents

Researchers should implement a systematic quality control approach, documenting all variables and conditions for each experiment to identify potential sources of variation.

How can SPAC1A6.11 antibody be adapted for high-throughput screening applications?

Adapting SPAC1A6.11 antibody for high-throughput applications requires methodological considerations:

• Assay miniaturization:

  • Optimize antibody concentrations for 384-well or 1536-well formats

  • Determine minimum detection limits in reduced volumes

  • Validate signal consistency across well positions

• Automation compatibility:

  • Formulate antibody dilutions stable for automated handling

  • Test robotic dispensing effects on antibody activity

  • Validate batch consistency across plates

• Readout optimization:

  • Develop fluorescence or luminescence-based detection

  • Calibrate signal dynamic range for screening windows

  • Implement Z'-factor analysis to assess assay quality

• Data analysis pipelines:

  • Establish normalization methods appropriate for antibody-based signals

  • Develop statistical approaches for hit identification

  • Implement quality control metrics for plate acceptance

• Validation strategies:

  • Incorporate positive and negative controls on each plate, including the provided control antigens

  • Develop secondary confirmation assays

  • Establish dose-response testing for identified hits

Contemporary high-throughput applications might employ machine learning approaches similar to those used in antibody development to analyze complex datasets generated with SPAC1A6.11 antibody.

What considerations are important when designing co-localization studies using SPAC1A6.11 antibody?

Co-localization studies require detailed methodological planning:

• Fixation optimization:

  • Test multiple fixatives (formaldehyde, methanol, etc.)

  • Determine optimal fixation duration and temperature

  • Validate antibody performance after each fixation method

• Antibody compatibility:

  • Ensure secondary antibodies don't cross-react

  • Select fluorophores with minimal spectral overlap

  • Establish sequential staining protocols if using multiple rabbit antibodies

• Microscopy parameters:

  • Optimize exposure settings to prevent saturation

  • Establish consistent acquisition parameters

  • Implement appropriate optical sectioning techniques

• Quantification approaches:

  • Select appropriate co-localization coefficients (Pearson's, Manders', etc.)

  • Establish thresholding criteria

  • Develop statistical analysis for biological replicates

• Controls:

  • Include known co-localizing proteins as positive controls

  • Use non-colocalizing proteins as negative controls

  • Employ pre-immune serum for background assessment

For yeast studies, the small cell size may necessitate super-resolution techniques for meaningful co-localization analysis with SPAC1A6.11 antibody.

How might SPAC1A6.11 antibody be incorporated into emerging single-cell analysis technologies?

Emerging single-cell technologies offer exciting possibilities for SPAC1A6.11 research:

• Mass cytometry (CyTOF) applications:

  • Metal-conjugated SPAC1A6.11 antibodies could enable multiplexed protein detection

  • Requires validation of metal conjugation effects on binding efficiency

  • Could reveal population heterogeneity in SPAC1A6.11 expression

• Single-cell proteomics:

  • Antibody-based enrichment prior to mass spectrometry

  • Target identification in complex single-cell lysates

  • Correlation of SPAC1A6.11 levels with other proteins at single-cell resolution

• Spatial transcriptomics integration:

  • Combining antibody detection with RNA localization

  • Correlating protein presence with gene expression in situ

  • Developing combined protocols for simultaneous detection

• Microfluidic applications:

  • Antibody immobilization in droplet-based assays

  • Single-cell western blotting using SPAC1A6.11 antibody

  • Development of yeast-compatible microfluidic devices

These emerging technologies would require significant protocol development but could provide unprecedented insights into SPAC1A6.11 function at the single-cell level, similar to recent advancements in antibody technology development .

What methodological approaches could enhance specificity when using SPAC1A6.11 antibody in complex experimental systems?

Advanced methods to enhance SPAC1A6.11 antibody specificity include:

• Epitope-specific purification:

  • Immobilize specific peptide epitopes for antibody subset isolation

  • Enrich antibodies targeting unique regions of SPAC1A6.11

  • Validate specificity of each epitope-specific fraction

• Competitive blocking strategies:

  • Develop peptide libraries for selective blocking

  • Identify minimal epitope sequences for specific blocking

  • Implement graduated blocking protocols

• CRISPR-based validation:

  • Generate epitope-tagged or epitope-modified variants

  • Create cell lines with specific mutations in antibody-binding regions

  • Validate antibody specificity across engineered cell lines

• Advanced bioinformatic approaches:

  • Implement epitope prediction algorithms

  • Map potential cross-reactivity using proteome-wide sequence analysis

  • Develop species-specific binding prediction models

• Combinatorial detection methods:

  • Pair antibody detection with orthogonal methods (MS, activity assays)

  • Implement multiplexed detection systems

  • Develop correlation metrics between detection methods

These approaches align with emerging trends in antibody technology, where computational methods enhance experimental design and validation .