SPAC17A2.10c Antibody

What is SPAC17A2.10c and why is it significant for research?

SPAC17A2.10c is an uncharacterized membrane protein found in Schizosaccharomyces pombe (fission yeast), which is a model organism widely used in molecular and cellular biology research . The protein is localized in both the cytoplasm and nucleus membrane as a multi-pass membrane protein. While its specific function remains largely uncharacterized, antibodies against this protein are valuable tools for studying membrane protein dynamics, yeast cellular processes, and protein-protein interactions in S. pombe. The significance lies in its potential role in membrane biology and cellular compartmentalization in this important model organism.

What are the common applications of SPAC17A2.10c antibody in fission yeast research?

The SPAC17A2.10c antibody serves multiple research purposes in S. pombe studies:

• Protein localization studies: Using immunofluorescence to determine subcellular distribution of the target protein

• Protein expression analysis: Western blotting to quantify protein levels across different experimental conditions

• Protein-protein interaction studies: Immunoprecipitation to identify binding partners

• Cell cycle regulation research: Examining protein expression changes during different phases

• Membrane protein dynamics: Studying trafficking and turnover of membrane proteins

What species reactivity and cross-reactivity can be expected with SPAC17A2.10c antibodies?

Based on available data, SPAC17A2.10c antibodies are primarily designed for specificity to Schizosaccharomyces pombe (strain 972 / ATCC 24843) . Cross-reactivity studies with other yeast species or higher eukaryotes have not been extensively documented. When using this antibody, researchers should expect:

• High specificity for S. pombe SPAC17A2.10c protein

• Potential cross-reactivity with highly conserved membrane proteins in closely related yeast species

• Limited to no cross-reactivity with mammalian cells or other distant organisms

For critical experiments, validation of specificity through knockout controls is recommended, particularly when applying these antibodies to species other than S. pombe .

What are the optimal conditions for using SPAC17A2.10c antibody in Western blotting?

For optimal Western blotting results with SPAC17A2.10c antibody, follow these methodological guidelines:

Sample preparation:

• Extract total protein from S. pombe using either glass bead lysis or enzymatic digestion

• Include protease inhibitors to prevent degradation of membrane proteins

• Use a membrane protein-compatible lysis buffer (containing 1-2% detergent such as Triton X-100)

Electrophoresis and transfer conditions:

• Use 10-12% SDS-PAGE gels for optimal resolution

• Transfer to PVDF membranes (preferred over nitrocellulose for membrane proteins)

• Use semi-dry transfer at 15V for 1 hour or wet transfer at 30V overnight at 4°C

Antibody incubation:

• Blocking: 5% non-fat milk in TBST, 1 hour at room temperature

• Primary antibody: 1:1000 dilution in 2% BSA/TBST, overnight at 4°C

• Secondary antibody: 1:5000 dilution in 2% BSA/TBST, 1 hour at room temperature

Detection:

• Enhanced chemiluminescence (ECL) substrate for standard detection

• Expected molecular weight: Based on the protein sequence, expect bands at the appropriate kDa range for SPAC17A2.10c

Note that membrane proteins can sometimes migrate at apparent molecular weights different from their calculated size due to hydrophobicity and post-translational modifications .

How can SPAC17A2.10c antibody be used effectively in immunofluorescence microscopy?

For successful immunofluorescence experiments with SPAC17A2.10c antibody in S. pombe, follow this protocol:

Cell fixation and permeabilization:

• Fix log-phase S. pombe cells with 3.7% formaldehyde for 30 minutes

• Digest cell wall with zymolyase (1mg/ml) for 30-60 minutes at 37°C

• Permeabilize with 0.1% Triton X-100 for 5 minutes

Antibody staining:

• Block with 3% BSA in PBS for 1 hour

• Incubate with SPAC17A2.10c antibody at 1:100-1:500 dilution overnight at 4°C

• Wash 3 times with PBS

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

• Counterstain with DAPI (1μg/ml) to visualize nuclei

Imaging considerations:

• Use confocal microscopy for optimal resolution of membrane structures

• Employ Z-stack imaging to capture the full distribution of the protein

• Include proper controls: secondary-only control and ideally a SPAC17A2.10c knockout strain

For membrane proteins like SPAC17A2.10c, careful optimization of permeabilization conditions is crucial to maintain membrane structure while allowing antibody access .

What approach should be used to validate SPAC17A2.10c antibody specificity?

Validation of antibody specificity is essential for reliable research results. For SPAC17A2.10c antibody, employ these validation approaches:

Genetic validation:

• Test the antibody on a SPAC17A2.10c gene deletion strain (negative control)

• Test on cells overexpressing SPAC17A2.10c (positive control)

Biochemical validation:

• Perform peptide competition assay

• Use recombinant SPAC17A2.10c protein as a positive control

• Perform immunoprecipitation followed by mass spectrometry to confirm target identity

Cross-validation:

• Compare results using different antibody clones (if available)

• Compare with localization of epitope-tagged versions of SPAC17A2.10c (e.g., GFP fusion)

The yeast two-hybrid approach can also be used for characterizing the antibody target, as described in related antibody research . This method can help confirm the specificity of the antibody binding domain and reveal potential cross-reactivity.

What are the common challenges when using SPAC17A2.10c antibody and how can they be addressed?

Working with antibodies against membrane proteins like SPAC17A2.10c presents several challenges:

For membrane proteins like SPAC17A2.10c, additionally consider using specialized detergents (e.g., CHAPS, DDM) that better preserve membrane protein structure while ensuring extraction efficiency .

How can the sensitivity of SPAC17A2.10c antibody be improved for detecting low abundance proteins?

To enhance detection sensitivity of low-abundance SPAC17A2.10c protein:

For Western blotting:

• Signal amplification: Use high-sensitivity chemiluminescent substrates (e.g., femto-level ECL)

• Sample concentration: Enrich membrane fractions through ultracentrifugation

• Loading optimization: Increase total protein loaded (up to 50-80µg per lane)

• Detection systems: Use digital imaging systems with high dynamic range instead of film

• Enhanced antibody binding: Use signal enhancer solutions before primary antibody incubation

For immunofluorescence:

• Tyramide signal amplification (TSA): Enzymatically deposits multiple fluorophores per antibody binding event

• Alternative fixation: Try methanol fixation which can sometimes better preserve certain epitopes

• Antigen retrieval: Apply mild heat treatment in citrate buffer prior to antibody incubation

• Microscopy optimization: Use high-sensitivity cameras and appropriate filter sets

• Sample preparation: Synchronize yeast cultures to capture peak expression phases

For quantitative applications, consider developing a sandwich ELISA using the SPAC17A2.10c antibody paired with another antibody recognizing a different epitope of the same protein, which can significantly improve sensitivity compared to single antibody detection methods .

How should storage and handling of SPAC17A2.10c antibody be optimized to maintain its activity?

Proper storage and handling significantly impact antibody performance over time:

Storage conditions:

• Store concentrated antibody stocks (>0.5 mg/ml) at -20°C or -80°C in small aliquots to avoid freeze-thaw cycles

• Working dilutions can be stored at 4°C with preservative (0.03% Proclin 300) for up to 2 weeks

• For long-term storage, adding glycerol to 50% can prevent damage from freeze-thaw cycles

Handling recommendations:

• Avoid protein denaturation by never vortexing antibody solutions (gentle mixing only)

• Centrifuge briefly before opening vials to collect solution at the bottom

• Use low protein-binding tubes and pipette tips for dilution

• Monitor for bacterial contamination (cloudiness or unusual odor)

• Document lot numbers and prepare standardized dilutions for experimental consistency

Stabilization additives:

• 50% glycerol, 0.01M PBS, pH 7.4 has been shown to maintain antibody activity

• For working solutions, adding 1% BSA or 5% glycerol can provide additional stability

• Avoid repeated freeze-thaw cycles; limit to maximum 5 cycles

Following these guidelines will help ensure consistent performance across experiments and maximize the usable lifetime of the antibody.

How can SPAC17A2.10c antibody be used in combination with yeast genetic techniques for functional studies?

Integrating antibody-based approaches with yeast genetics provides powerful insights into SPAC17A2.10c function:

Combining with gene deletion/mutation approaches:

• Create SPAC17A2.10c point mutations or domain deletions in S. pombe

• Use the antibody to assess changes in protein localization, abundance, or interaction partners

• Determine structure-function relationships by correlating mutant phenotypes with antibody-detected changes

Integration with yeast two-hybrid (Y2H) systems:
The antibody can validate Y2H interactions by:

• Confirming interaction partners through co-immunoprecipitation

• Verifying subcellular co-localization of interaction partners

• Analyzing competition between antibody binding and protein-protein interactions

Combined with yeast surface display:

• Generate yeast display libraries of SPAC17A2.10c variants

• Use the antibody for flow cytometry screening of binding properties

• Isolate high-affinity binding variants for structure-function studies

This integrated approach has been effectively used in similar research contexts to characterize antibody targets through yeast two-hybrid methods and could be applied to SPAC17A2.10c studies . The yeast display methodology in particular allows rapid screening of protein variants and epitope mapping as demonstrated in related research .

What approaches can be used to identify the epitope recognized by SPAC17A2.10c antibody?

Epitope mapping is critical for understanding antibody specificity and function. For SPAC17A2.10c antibody, these methods can be employed:

Fragment-based epitope mapping:

• Generate overlapping fragments of SPAC17A2.10c protein

• Express fragments in bacteria or yeast display systems

• Test antibody binding to each fragment by ELISA or flow cytometry

• Narrow down to minimal binding region through nested deletions

Peptide array analysis:

• Synthesize overlapping peptides (15-20 amino acids) spanning SPAC17A2.10c sequence

• Spot peptides on membrane or glass slide

• Probe with the antibody followed by secondary detection

• Identify reactive peptides that represent linear epitopes

Site-directed mutagenesis:

• Based on initial mapping, create point mutations in potential epitope regions

• Test antibody binding to mutants by ELISA or Western blot

• Identify critical residues for antibody recognition

Structural approaches:

• X-ray crystallography of antibody-peptide complexes

• Hydrogen-deuterium exchange mass spectrometry to identify protected regions

This methodology has been successfully applied in studies such as those identifying epitopes for Sp17 and SpA5 antibodies, as referenced in the search results , and could be adapted for SPAC17A2.10c antibody characterization.

How can SPAC17A2.10c antibody be utilized in high-throughput screening or proteomics workflows?

The SPAC17A2.10c antibody can be integrated into advanced screening and proteomics approaches:

Antibody microarray applications:

• Immobilize SPAC17A2.10c antibody on microarray slides

• Probe with differentially labeled protein samples from various conditions

• Detect changes in SPAC17A2.10c levels or modifications across conditions

High-content screening:

• Use the antibody in automated immunofluorescence workflows

• Screen genetic or chemical libraries for effects on SPAC17A2.10c localization

• Identify factors that regulate membrane protein trafficking

Mass spectrometry-based proteomics:

• Immunoprecipitate SPAC17A2.10c and associated proteins

• Analyze by LC-MS/MS to identify interaction partners

• Quantify changes in the SPAC17A2.10c "interactome" under different conditions

In vivo biotinylation for biomarker discovery:
Similar to the approach described in search result , the antibody could be:

• In vivo biotinylated to create "biobodies"

• Used to immunoprecipitate target and associated proteins

• Applied in protein complex identification through mass spectrometry

This approach is particularly valuable for membrane proteins like SPAC17A2.10c where traditional interaction methods may be more challenging due to hydrophobicity and complex membrane environments.

What methodologies can be used to develop improved versions of SPAC17A2.10c antibodies?

For researchers seeking to develop enhanced SPAC17A2.10c antibodies, several advanced engineering approaches can be employed:

Affinity maturation through display technologies:

• Create antibody fragment libraries with mutations in complementarity-determining regions (CDRs)

• Display libraries on yeast or phage surface

• Select high-affinity variants through increasing stringency of binding conditions

• Convert selected variants to full antibodies

Antibody humanization/optimization:

• Analyze framework regions for potential immunogenicity

• Modify framework while preserving CDRs

• Test engineered variants for improved stability and reduced aggregation

Format optimization:

• Convert to different antibody formats (Fab, scFv, nanobody) for specific applications

• Develop bispecific antibodies targeting SPAC17A2.10c and another protein of interest

• Create antibody-drug conjugates for targeted protein degradation studies

High-throughput functional screening:
As demonstrated in search result , autonomous hypermutation in yeast can rapidly generate potent antibody variants:

• Encode antibody sequence in specially designed yeast vectors

• Allow continuous diversification through error-prone DNA polymerases

• Select improved variants based on binding or functional assays

This approach resulted in ~20-fold functional affinity enhancement in similar antibody engineering efforts and could be applied to develop improved SPAC17A2.10c antibodies for specific research applications.

How does SPAC17A2.10c antibody performance compare with antibodies against other yeast membrane proteins?

When evaluating SPAC17A2.10c antibody performance against other yeast membrane protein antibodies, researchers should consider these comparative aspects:

For comprehensive studies, researchers often employ antibodies against established membrane protein markers (e.g., ER, Golgi, plasma membrane) alongside SPAC17A2.10c antibody for co-localization studies. This comparative approach helps establish the relative subcellular distribution pattern of the target protein .

What are the considerations for using SPAC17A2.10c antibody in non-model yeast species or other fungi?

When extending SPAC17A2.10c antibody use beyond S. pombe:

Sequence homology analysis:

• Perform sequence alignment of SPAC17A2.10c with homologs in target species

• Focus on conservation in the epitope region (if known)

• Predict cross-reactivity based on percent identity in key regions

Validation requirements:

• Western blots with positive controls (S. pombe extract)

• Include negative controls (non-expressing cells/species)

• Consider epitope-tagged versions of homologous proteins as validation

Optimization strategies:

• Increase antibody concentration (typically 2-5× higher than for S. pombe)

• Extend incubation times (overnight at 4°C recommended)

• Modify extraction buffers to account for different cell wall composition

• Adjust permeabilization protocols for species-specific differences

Species-specific challenges:

• Cell wall thickness varies across species, affecting antibody penetration

• Post-translational modifications may differ, altering epitope accessibility

• Expression levels of homologs may be substantially different

While homologs exist across fungi, the uncharacterized nature of SPAC17A2.10c means that cross-species applications should be approached with caution and thorough validation.

How can SPAC17A2.10c antibody be used to study protein-protein interactions in membrane complexes?

For investigating membrane protein interactions involving SPAC17A2.10c:

Co-immunoprecipitation optimization for membrane proteins:

• Use mild detergents (0.5-1% NP-40, Digitonin, or CHAPS) to preserve interactions

• Include chemical crosslinking step (e.g., DSP, formaldehyde) to stabilize transient interactions

• Consider native extraction conditions to maintain protein complexes

• Perform parallel experiments with and without crosslinking

Proximity-based interaction methods:

• Proximity ligation assay (PLA):

  • Detect SPAC17A2.10c interactions with candidate proteins in situ

  • Requires antibodies against both interaction partners from different species

  • Generates fluorescent signal only when proteins are within 40nm

• BioID or TurboID proximity labeling:

  • Fuse biotin ligase to SPAC17A2.10c

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • Particularly useful for transient or weak interactions in membrane environments

Validation approaches:

• Reverse co-immunoprecipitation with antibodies against interaction partners

• Genetic perturbation (deletion/overexpression) of one partner

• Functional assays to demonstrate biological relevance of interactions