SPAC22H10.11c Antibody

What is SPAC22H10.11c and why is it significant for S. pombe research?

SPAC22H10.11c is a protein found in Schizosaccharomyces pombe (fission yeast) with UniProt accession number Q10304 . While detailed functional characterization is still developing, antibodies against this protein serve as valuable tools for studying protein expression, localization, and interactions in S. pombe. This protein is particularly significant for researchers exploring the molecular biology of fission yeast as a model organism. When designing experiments, researchers should consider both the specific isoform and cellular compartment in which the protein may be expressed.

What applications are most suitable for SPAC22H10.11c antibody?

The SPAC22H10.11c antibody has been validated for ELISA and Western Blot (WB) applications . For Western Blot applications, researchers should optimize protein extraction protocols specific to fission yeast, considering the rigid cell wall that requires specialized lysis procedures. The antibody's polyclonal nature makes it particularly suitable for immunoprecipitation experiments where detection of native conformational epitopes may be advantageous. Additionally, researchers might explore adapting protocols for immunofluorescence or chromatin immunoprecipitation, though these would require custom validation.

How should SPAC22H10.11c antibody be stored to maintain optimal activity?

The antibody should be stored at -20°C or -80°C upon receipt . Critically, researchers should avoid repeated freeze-thaw cycles as they can significantly compromise antibody activity through protein denaturation and aggregation. For laboratories conducting long-term studies, consider aliquoting the antibody upon receipt into single-use volumes. The storage buffer (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) is designed to maintain stability during freezing . When calculating experimental timelines, account for the antibody's optimal activity window after reconstitution (typically 6-12 months when properly stored).

What controls are essential when using SPAC22H10.11c antibody for Western blot experiments?

When analyzing data, each experimental replicate should include the complete set of controls. Additionally, researchers should conduct a titration series (ranging from 1:500 to 1:5000 dilutions) to determine optimal antibody concentration for their specific experimental system, as background signals in S. pombe extracts can vary based on growth conditions and extraction methods.

How can I validate SPAC22H10.11c antibody specificity for my research?

Validation should include multiple complementary approaches. First, compare immunoblotting patterns between wild-type strains and strains where SPAC22H10.11c has been genetically deleted or modified (such as through CRISPR-Cas9). Second, utilize epitope-tagged SPAC22H10.11c constructs expressed in S. pombe and compare detection with both anti-tag antibodies and the SPAC22H10.11c antibody. Third, perform mass spectrometry analysis of immunoprecipitated material to confirm the presence of SPAC22H10.11c and identify potential cross-reactive proteins. Finally, implement a peptide competition assay using the immunizing antigen to demonstrate binding specificity. These approaches collectively provide a robust validation framework that addresses both false positives and false negatives.

What are the key considerations when designing immunoprecipitation experiments with this antibody?

As a polyclonal antibody affinity-purified against the recombinant SPAC22H10.11c protein , this reagent presents specific considerations for immunoprecipitation. First, determine the optimal antibody-to-bead ratio through titration experiments (typically 2-10 μg antibody per 20-50 μl of beads). Second, evaluate both native and denaturing extraction conditions, as the polyclonal nature may recognize multiple epitopes with different accessibility in folded versus unfolded states. Third, optimize wash stringency to balance between preserving specific interactions and eliminating background. Fourth, consider crosslinking the antibody to beads to prevent antibody co-elution with the target protein. Finally, validate results using reciprocal IP approaches with known interaction partners or alternative antibodies when available.

How can I adapt SPAC22H10.11c antibody for immunofluorescence microscopy in fission yeast?

While the antibody is primarily validated for ELISA and Western blot applications , adapting it for immunofluorescence requires specific methodological considerations. First, optimize fixation methods, comparing paraformaldehyde (preserves structure but may mask epitopes), methanol (better for some intracellular antigens), and hybrid approaches. Second, test varying cell wall digestion protocols using enzymes like zymolyase or lysing enzymes at different concentrations and incubation times. Third, implement a blocking optimization matrix testing BSA (1-5%), normal serum (5-10%), and non-ionic detergents (0.1-0.3% Triton X-100) to reduce background. Fourth, determine optimal antibody concentration through a titration series (1:50 to 1:500). Fifth, validate specificity using SPAC22H10.11c deletion strains as negative controls. Document all parameters systematically to establish a reproducible protocol.

What approaches can address epitope masking issues when the SPAC22H10.11c protein is in complex with other molecules?

Epitope masking presents a significant challenge when studying proteins in their native complexes. First, implement multiple extraction conditions comparing gentle non-ionic detergents (NP-40, Triton X-100) with more stringent ionic detergents (SDS, deoxycholate) to disrupt different levels of protein interactions. Second, test various epitope retrieval methods including heat treatment, pH shifts (citrate buffer pH 6.0 or Tris-EDTA pH 9.0), and controlled proteolytic digestion. Third, consider proximity labeling approaches (BioID, APEX) as complementary methods to detect associations without relying on direct antibody access to epitopes. Fourth, use crosslinking mass spectrometry to map interaction interfaces and predict potential epitope masking. Finally, consider generating additional antibodies targeting different regions of SPAC22H10.11c if persistent masking issues occur.

How can I quantitatively assess post-translational modifications of SPAC22H10.11c using this antibody?

To investigate post-translational modifications (PTMs), implement a multi-layered approach. First, perform immunoprecipitation with the SPAC22H10.11c antibody followed by Western blotting with modification-specific antibodies (phospho, ubiquitin, SUMO, etc.). Second, use phosphatase or deubiquitinase treatments on parallel samples to confirm modification specificity. Third, combine with Phos-tag™ SDS-PAGE to resolve phosphorylated species with mobility shifts. Fourth, employ 2D gel electrophoresis (separating by isoelectric point and molecular weight) to resolve modified protein populations. Finally, confirm findings using mass spectrometry analysis of immunoprecipitated material with specific attention to fragmentation patterns characteristic of PTMs. When interpreting data, consider the polyclonal nature of the antibody , which may have differential affinity for various modified forms of the protein.

What strategies can resolve inconsistent SPAC22H10.11c detection in Western blot applications?

For reproducible results, implement a systematic laboratory record that documents all variables including culture conditions (temperature, media composition, growth phase), extraction methods (buffer composition, mechanical disruption parameters), and detection settings (exposure times, imaging parameters). This approach enables identification of subtle technical variables that may affect experimental outcomes.

How can I address potential cross-reactivity with other Schizosaccharomyces proteins?

Cross-reactivity represents a significant concern for polyclonal antibodies. First, perform bioinformatic analysis to identify S. pombe proteins with sequence homology to SPAC22H10.11c. Second, express and purify these potential cross-reactive proteins for competitive binding assays. Third, utilize gene deletion libraries to systematically test reactivity patterns in strains lacking potential cross-reactive proteins. Fourth, implement a phosphopeptide mapping approach combined with mass spectrometry to conclusively identify all proteins recognized by the antibody in complex samples. Fifth, consider pre-adsorption of the antibody with cellular extracts from SPAC22H10.11c deletion strains to remove antibodies targeting conserved epitopes. Document cross-reactivity and incorporate this information into experimental design and data interpretation.

What factors might contribute to lot-to-lot variability when using this polyclonal antibody?

Polyclonal antibodies inherently show lot-to-lot variability due to their production in animals. Key factors include: first, differences in animal immune responses leading to variable epitope recognition profiles; second, variations in affinity purification efficiency affecting antibody concentration and specificity; third, potential differences in antibody subclass distributions affecting binding characteristics; fourth, variable stability during storage and shipping conditions. To mitigate these issues, researchers should: (1) purchase sufficient quantity from a single lot for entire study duration, (2) perform standardized validation for each new lot, (3) maintain reference samples from previous successful experiments, and (4) document lot numbers in all publications and reports to facilitate reproducibility assessment.

How does the performance of SPAC22H10.11c antibody compare with other fission yeast protein detection methods?

When evaluating detection methodologies, researchers should consider multiple parameters. Direct protein tagging (GFP, FLAG, HA tags) offers live-cell visualization capabilities but may interfere with protein function or localization. Mass spectrometry provides unbiased detection with quantitative capabilities but requires specialized equipment and expertise. The SPAC22H10.11c antibody approach offers detection of the native, unmodified protein but depends on epitope accessibility and antibody specificity . Proximity-based labeling methods (BioID, APEX) offer interaction detection advantages but may identify false positives. RNA-based methods (qRT-PCR, RNA-seq) measure transcript rather than protein levels. Each approach has distinct advantages for specific research questions, and complementary application of multiple methods provides the most robust experimental framework.

What methodological adaptations are needed when comparing SPAC22H10.11c expression across different S. pombe strains or genetic backgrounds?

Cross-strain comparisons require careful methodological controls. First, standardize growth conditions (temperature, media, growth phase) to minimize physiological variation. Second, implement internal loading controls appropriate for the genetic backgrounds being studied (validate that chosen housekeeping genes maintain consistent expression across strains). Third, prepare standard curves using purified recombinant SPAC22H10.11c for absolute quantification rather than relying solely on relative measurements. Fourth, consider genetic background effects on protein extraction efficiency by comparing multiple extraction methods across strains. Fifth, account for potential strain-specific post-translational modifications by implementing approaches like Phos-tag™ gels or 2D electrophoresis. These methodological controls enable meaningful interpretation of expression differences independent of technical artifacts.

How can SPAC22H10.11c antibody be integrated with other research techniques for comprehensive protein function analysis?

A multi-method approach provides the most comprehensive understanding. First, combine SPAC22H10.11c antibody-based detection with genetic approaches (deletion, overexpression, mutation) to correlate protein levels with phenotypic consequences. Second, integrate with structural biology techniques (X-ray crystallography, cryo-EM) to connect detected expression patterns with structural information. Third, complement with functional genomics (synthetic genetic arrays, CRISPR screens) to identify genetic interactions. Fourth, incorporate computational biology approaches (homology modeling, network analysis) to predict potential functions based on detected expression patterns. Fifth, implement temporal studies (cell cycle synchronization, developmental time courses) to map dynamic expression changes. This integrated approach connects static antibody-generated data with dynamic functional insights.

What protocol modifications are needed for efficient SPAC22H10.11c detection in different cellular fractions?

Detecting SPAC22H10.11c in different cellular compartments requires fraction-specific optimizations:

For each fraction, implement Western blot controls using fraction-specific marker proteins (e.g., tubulin for cytoplasm, histone H3 for chromatin). Additionally, assess cross-contamination between fractions through these markers to ensure isolation purity. This fractionation approach enables detailed investigation of SPAC22H10.11c subcellular distribution and potential compartment-specific functions.

What is the optimal immunoprecipitation protocol for studying SPAC22H10.11c protein interactions?

For optimal immunoprecipitation of SPAC22H10.11c and associated proteins, follow this methodological framework: First, evaluate cell lysis conditions comparing mechanical disruption (bead beating) with enzymatic approaches (lysing enzymes followed by osmotic lysis) to determine optimal protein extraction while preserving interactions. Second, test multiple buffer compositions, comparing low stringency (150mM NaCl, 0.5% NP-40) with medium stringency (250mM NaCl, 0.1% SDS, 0.5% deoxycholate) conditions. Third, optimize antibody concentration (2-10μg per reaction) and incubation parameters (4°C overnight vs. room temperature for 2 hours). Fourth, compare different immobilization matrices (Protein A/G, direct coupling to activated resins) for capture efficiency. Fifth, develop an elution strategy comparing harsh conditions (SDS, low pH) with more specific approaches (competing peptides). Finally, analyze immunoprecipitated material using both targeted (Western blot) and unbiased (mass spectrometry) methods to comprehensively characterize the interaction network.

How can I develop a quantitative ELISA assay for SPAC22H10.11c using this antibody?

Developing a quantitative ELISA requires systematic optimization. First, determine optimal coating conditions by testing different SPAC22H10.11c antibody concentrations (0.5-10 μg/mL) and coating buffers (carbonate/bicarbonate pH 9.6 vs. PBS pH 7.4). Second, establish blocking parameters by comparing BSA (1-5%), casein (1-2%), and commercial blocking reagents with overnight incubation at 4°C. Third, optimize sample preparation through testing various lysis methods (sonication, detergent extraction) and determining the linear detection range (create standard curves with recombinant SPAC22H10.11c). Fourth, determine detection antibody parameters, potentially using a secondary detection antibody targeting a different epitope for sandwich ELISA format. Fifth, compare colorimetric, fluorescent, and chemiluminescent detection systems for optimal sensitivity and dynamic range. Document all optimization steps to establish a reproducible quantitative assay with defined sensitivity, specificity, and linear detection range parameters.