SPAC18B11.08c Antibody

What is SPAC18B11.08c and why is it studied in fission yeast?

SPAC18B11.08c refers to an uncharacterized protein in Schizosaccharomyces pombe (strain 972/24843), commonly known as fission yeast. While the specific function of this protein remains largely unknown, it is studied as part of broader investigations into the S. pombe proteome. Fission yeast serves as an important model organism in molecular and cellular biology research due to its relatively simple genome and cellular processes that share similarities with higher eukaryotes.

The study of uncharacterized proteins like SPAC18B11.08c contributes to our fundamental understanding of eukaryotic cell biology. These investigations typically aim to determine protein localization, interaction partners, and potential roles in cellular processes such as cell division, DNA replication, or stress responses. S. pombe has proven particularly valuable for understanding cell cycle regulation and chromosomal dynamics, as demonstrated in recent studies of mating-type switching mechanisms .

What are the key specifications of the SPAC18B11.08c antibody?

The SPAC18B11.08c antibody is a rabbit-derived polyclonal antibody specifically raised against Schizosaccharomyces pombe (strain 972/24843) protein. Its key specifications include:

The antibody has been specifically designed to recognize epitopes of the SPAC18B11.08c protein and has undergone purification through antigen-affinity methods to ensure specificity . This purification process helps minimize cross-reactivity with other proteins, though as with all polyclonal antibodies, batch-to-batch variation may occur.

What validated applications can the SPAC18B11.08c antibody be used for?

Based on available information, the SPAC18B11.08c polyclonal antibody has been validated for the following research applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection and measurement of the SPAC18B11.08c protein in various sample preparations. This method is particularly useful for determining protein concentration and studying expression levels under different conditions.

• Western Blot (WB): For identification and semi-quantitative analysis of the protein in cell lysates. This technique allows researchers to confirm the presence of the protein, estimate its molecular weight, and detect potential post-translational modifications .

These applications make the antibody suitable for various research contexts, including:

• Protein expression studies in different growth conditions or mutant strains

• Verification of genetic knockout experiments

• Investigation of protein regulation during cell cycle progression or stress responses

• Subcellular localization studies when combined with fractionation techniques

While not explicitly mentioned in the validation data, similar polyclonal antibodies may also be applicable for immunoprecipitation (IP) studies, though additional validation would be required before employing the antibody for such purposes.

How can the SPAC18B11.08c antibody be optimized for studying mating-type switching in fission yeast?

Mating-type switching in S. pombe involves complex molecular mechanisms including the Swi2-Swi5 complex and its interaction with various cellular components. The SPAC18B11.08c antibody could be utilized to investigate potential connections between this uncharacterized protein and mating-type switching machinery through several optimized approaches:

• Co-immunoprecipitation studies: The antibody could be used to pull down SPAC18B11.08c and identify whether it interacts with known components of the mating-type switching apparatus, such as the Swi2-Swi5 complex. Recent research has shown that Swi2 contains functionally important motifs including a Swi6-binding site and DNA-binding AT-hooks that are crucial for donor selection during mating-type switching .

• Chromatin immunoprecipitation (ChIP): If SPAC18B11.08c is suspected to associate with chromatin, researchers could use the antibody in ChIP experiments to determine if it localizes to specific chromosomal regions, particularly those involved in mating-type determination, such as the mat1, mat2-P, or mat3-M loci .

• Cell-type specific expression analysis: Western blot analysis using the SPAC18B11.08c antibody could be performed on P and M cell types separately to detect potential differences in expression levels or post-translational modifications that might correlate with cell type.

A methodological approach might include:

• Synchronizing yeast cultures to study protein expression changes during mating-type switching events

• Comparing protein localization between wild-type and switching-deficient mutants

• Analyzing potential association with recombination enhancers (SRE2 and SRE3) that are known to be important in donor selection

What experimental design considerations are important when using the SPAC18B11.08c antibody for co-immunoprecipitation studies?

When designing co-immunoprecipitation (Co-IP) experiments with the SPAC18B11.08c antibody, researchers should consider several critical factors:

• Lysis conditions optimization:

  • S. pombe has a rigid cell wall requiring effective disruption methods, typically using glass beads or enzymatic approaches

  • Buffer composition should preserve protein-protein interactions while enabling efficient cell lysis

  • Include protease inhibitors to prevent degradation during sample preparation

  • Consider crosslinking agents for capturing transient interactions

• Antibody binding strategy:

  • Direct method: Add SPAC18B11.08c antibody to lysate, followed by Protein A/G beads

  • Pre-coupling method: Immobilize antibody on beads before adding to lysate

  • Determine optimal antibody:protein ratio through titration experiments

• Controls and validation:

  • Input control: Analyze a portion of pre-cleared lysate to confirm target protein presence

  • Negative control: Use non-specific IgG from the same species

  • Specificity control: Include samples from SPAC18B11.08c deletion strains if available

  • Reciprocal Co-IP: Confirm interactions by reversing the antibody used for pulldown

• Interaction verification methods:

  • Western blot analysis with antibodies against suspected interaction partners

  • Mass spectrometry for unbiased identification of co-precipitated proteins

  • Functional assays to confirm biological relevance of identified interactions

A robust experimental approach might involve comparing Co-IP results between normal growth conditions and specific cellular states, such as different cell cycle phases or stress conditions, to identify context-dependent interactions that might reveal SPAC18B11.08c function.

How can researchers investigate potential post-translational modifications of SPAC18B11.08c?

Investigation of post-translational modifications (PTMs) of SPAC18B11.08c requires specialized approaches beyond standard antibody applications:

• Electrophoretic mobility shift analysis:

  • Run parallel samples on standard SDS-PAGE gels to detect potential molecular weight changes indicative of modifications

  • Employ Phos-tag™ acrylamide gels specifically designed to retard the migration of phosphorylated proteins

  • Compare migration patterns before and after treatment with phosphatases or other modification-removing enzymes

• Specialized immunoprecipitation strategies:

  • Perform immunoprecipitation with the SPAC18B11.08c antibody under conditions that preserve specific modifications

  • For phosphorylation studies, include phosphatase inhibitors in all buffers

  • For ubiquitination analysis, use denaturing conditions to preserve ubiquitin conjugates

• Mass spectrometry approaches:

  • After immunoprecipitation with the SPAC18B11.08c antibody, analyze samples by LC-MS/MS

  • Employ specific fragmentation methods optimized for PTM detection

  • Use targeted approaches to look for particular modifications based on sequence analysis

• Integration with biological contexts:

  • Study PTM dynamics during cell cycle progression, as many S. pombe proteins undergo cell cycle-dependent modifications

  • Examine modifications in response to stress conditions that might reveal functional roles

  • Compare modification patterns in wild-type versus mutant strains defective in specific PTM pathways

When integrating these approaches, researchers should carefully document experimental conditions and include appropriate controls to distinguish genuine modifications from artifacts.

What is the optimal protocol for Western blot analysis using the SPAC18B11.08c antibody?

An optimized Western blot protocol for SPAC18B11.08c detection should account for the specific characteristics of S. pombe samples:

• Sample preparation:

  • Harvest 10-20 ml of S. pombe culture at mid-log phase (OD600 ~0.5-0.8)

  • Lyse cells using glass bead disruption in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail

  • Clear lysate by centrifugation at 14,000 × g for 15 minutes at 4°C

  • Quantify protein concentration using Bradford or BCA assay

• Gel electrophoresis and transfer:

  • Load 20-50 μg of total protein per lane on 10-12% SDS-PAGE gels

  • Include appropriate molecular weight markers

  • Transfer to PVDF membrane at 100V for 1 hour in cold transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol)

  • Verify transfer efficiency using reversible protein stain (e.g., Ponceau S)

• Antibody incubation and detection:

  • Block membrane with 5% non-fat dry milk in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature

  • Incubate with SPAC18B11.08c antibody at 1:1000 dilution in 5% BSA/TBST overnight at 4°C

  • Wash 3 × 10 minutes with TBST

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

  • Wash 3 × 10 minutes with TBST

  • Develop using enhanced chemiluminescence (ECL) substrate and image

• Controls and validation:

  • Positive control: Lysate from wild-type S. pombe

  • Negative control: Lysate from SPAC18B11.08c deletion strain if available

  • Loading control: Probe for housekeeping protein (e.g., α-tubulin or GAPDH)

  • Size verification: Confirm that detected band appears at expected molecular weight

This protocol should be optimized for each laboratory's specific conditions, with particular attention to antibody dilution and incubation times.

What are the critical validation steps for confirming SPAC18B11.08c antibody specificity?

Rigorous validation of the SPAC18B11.08c antibody is essential for generating reliable research data. Key validation steps include:

• Genetic validation approaches:

  • Test the antibody on samples from SPAC18B11.08c deletion strains (should show no signal)

  • Examine strains expressing tagged versions (e.g., GFP-tagged SPAC18B11.08c) and perform parallel detection with anti-tag antibodies

  • Analyze samples from strains with SPAC18B11.08c overexpression to confirm proportional signal increase

• Biochemical validation methods:

  • Perform peptide competition assays where the antibody is pre-incubated with the immunizing peptide (should eliminate specific signal)

  • Conduct Western blots under different conditions to confirm consistent detection

  • Verify that the detected protein appears at the expected molecular weight

• Cross-reactivity assessment:

  • Test on lysates from related yeast species to evaluate specificity

  • If closely related proteins are known, examine potential cross-reactivity

  • Consider testing in fractionated samples to evaluate non-specific binding to subcellular components

• Application-specific validation:

  • For each intended application (Western blot, ELISA), establish specific validation criteria

  • Document optimal conditions, dilutions, and expected results

  • Include appropriate positive and negative controls in each experiment

A comprehensive validation approach provides confidence in the antibody's specificity and reliability across different experimental contexts.

How can researchers combine the SPAC18B11.08c antibody with other techniques for comprehensive protein characterization?

Integrating the SPAC18B11.08c antibody with complementary techniques provides more robust characterization:

• Combining with imaging approaches:

  • Use immunofluorescence with the antibody to determine subcellular localization

  • Compare with live-cell imaging of fluorescently tagged SPAC18B11.08c

  • Consider super-resolution microscopy for detailed localization studies

  • Perform co-localization analysis with markers of cellular compartments

• Integration with omics approaches:

  • Use antibody detection to validate proteomics findings

  • Compare protein expression (via Western blot) with transcriptional data

  • Enrich samples via immunoprecipitation before mass spectrometry analysis

  • Correlate protein levels with phenotypic data from genetic screens

• Combination with genetic manipulation techniques:

  • Use the antibody to confirm knockout efficiency in deletion strains

  • Verify expression levels in conditional mutants

  • Analyze protein dynamics in strains with modified regulatory elements

  • Study protein-protein interactions through co-immunoprecipitation coupled with genetic perturbations

• Functional studies integration:

  • Correlate protein levels with phenotypic assays (e.g., growth rate, stress resistance)

  • Analyze protein behavior during specific cellular processes like mating-type switching

  • Combine with biochemical assays to test enzymatic activities if relevant

  • Use for directed proteomics in different cellular states or genetic backgrounds

A strategic experimental design might incorporate the antibody at multiple levels: initial detection and quantification via Western blot, localization studies via immunofluorescence, interaction analysis via co-IP, and validation of findings from high-throughput approaches.

What are common challenges when using the SPAC18B11.08c antibody and how can they be addressed?

Researchers working with the SPAC18B11.08c antibody may encounter several technical challenges:

• High background signal in Western blots:

  • Issue: Non-specific binding leading to multiple bands or smears

  • Solutions:

    • Increase blocking time/concentration (try 5% BSA instead of milk)

    • Optimize antibody dilution (try higher dilutions)

    • Increase washing stringency (add 0.1% SDS to TBST)

    • Use alternative blocking agents (casein, commercial blocking buffers)

    • Pre-absorb antibody with non-specific proteins

• Weak or no signal detection:

  • Issue: Insufficient antibody binding or protein abundance

  • Solutions:

    • Decrease antibody dilution (use more concentrated antibody)

    • Increase protein loading (50-100 μg per lane)

    • Extend exposure time for detection

    • Try more sensitive detection systems

    • Optimize transfer conditions (time, buffer composition)

    • Verify protein expression using alternative methods

• Inconsistent results between experiments:

  • Issue: Variable performance across different experiments

  • Solutions:

    • Standardize protein extraction and quantification methods

    • Aliquot antibody to avoid freeze-thaw cycles

    • Include consistent positive controls

    • Document and maintain consistent experimental conditions

    • Consider antibody storage conditions and shelf-life

• Issues with immunoprecipitation efficiency:

  • Issue: Poor protein recovery in pull-down experiments

  • Solutions:

    • Optimize antibody:bead:lysate ratios

    • Adjust binding conditions (time, temperature, buffer composition)

    • Consider crosslinking approaches

    • Test different bead types (magnetic vs. agarose)

    • Evaluate alternative lysis methods that better preserve epitopes

For each challenge, systematic optimization and careful documentation of conditions are essential for developing robust protocols.

How should researchers approach quantitative analysis of Western blot data generated with the SPAC18B11.08c antibody?

Quantitative analysis of Western blots requires careful attention to methodology and appropriate statistical approaches:

• Image acquisition and densitometry:

  • Use digital imaging systems rather than film for better dynamic range

  • Ensure exposure times avoid signal saturation

  • Perform densitometry using software like ImageJ, Image Studio, or specialized Western blot analysis programs

  • Define consistent region-of-interest selection methods

• Normalization strategies:

  • Always normalize to appropriate loading controls (e.g., tubulin, GAPDH)

  • Verify that loading controls remain stable across experimental conditions

  • Consider total protein normalization methods (Ponceau S, SYPRO Ruby) as alternatives

  • For complex experiments, include internal calibration standards

• Statistical analysis approaches:

  • For comparing two conditions:

    • Student's t-test (parametric) if data is normally distributed

    • Mann-Whitney U test (non-parametric) for non-normal distributions

  • For multiple conditions:

    • One-way ANOVA with post-hoc tests (Tukey or Bonferroni)

    • Consider repeated measures approaches for related samples

• Data presentation and reporting:

  • Present data as mean ± standard deviation or standard error

  • Include all biological replicates (minimum n=3)

  • Show representative Western blot images alongside quantitative graphs

  • Report exact p-values, not just significance thresholds

  • Distinguish between biological and technical replicates

A robust quantitative analysis should include:

• Clear description of normalization method

• Appropriate statistical tests with justification

• Presentation of both raw and normalized data where possible

• Consideration of both statistical and biological significance

What factors should be considered when comparing results from different batches of the SPAC18B11.08c polyclonal antibody?

Polyclonal antibodies inherently show batch-to-batch variation, requiring careful consideration when comparing results:

• Batch characterization and documentation:

  • Test each new batch alongside the previous batch

  • Document key performance metrics (optimal dilution, signal:noise ratio)

  • Create and maintain standard positive control samples for comparison

  • Record lot numbers and acquisition dates

• Calibration approaches:

  • Include standard samples of known concentration in each experiment

  • Generate batch-specific standard curves when quantitative analysis is required

  • Consider creating laboratory reference standards for long-term studies

  • Document relative sensitivities between batches

• Experimental design considerations:

  • Complete experimental series with a single antibody batch when possible

  • If batch changes are unavoidable mid-experiment, include overlapping samples

  • For longitudinal studies, create and freeze reference samples

  • Consider alternative detection methods for critical findings

• Data normalization strategies:

  • Normalize to batch-specific standards

  • Consider expressing results as percent change rather than absolute values

  • Use ratio measurements rather than raw intensities when appropriate

  • Employ statistical methods that account for batch effects (e.g., mixed-effects models)

Maintaining detailed documentation of antibody performance characteristics enables more reliable cross-batch comparisons and helps distinguish genuine biological variation from technical artifacts.

How might the SPAC18B11.08c antibody be used to investigate potential roles of this protein in DNA replication or repair pathways?

Given that many uncharacterized S. pombe proteins play roles in genome maintenance pathways, researchers could use the SPAC18B11.08c antibody to investigate potential functions in DNA replication or repair through several approaches:

• Cell cycle-dependent expression and localization analysis:

  • Synchronize S. pombe cultures and collect samples at defined cell cycle stages

  • Use the antibody to track SPAC18B11.08c protein levels by Western blot

  • Perform chromatin fractionation to determine association with DNA during different cell cycle phases

  • Combine with immunofluorescence to visualize potential co-localization with replication factories

• Response to DNA damage and replication stress:

  • Treat cells with DNA damaging agents (e.g., MMS, UV, hydroxyurea)

  • Analyze changes in protein levels, modifications, or localization using the antibody

  • Compare with known DNA replication checkpoint components such as those in the Rad3 pathway

  • Look for potential recruitment to sites of DNA damage

• Interaction studies with known replication/repair factors:

  • Perform co-immunoprecipitation with the SPAC18B11.08c antibody after replication stress

  • Analyze potential interactions with components of replication machinery

  • Consider reciprocal co-IPs with antibodies against Swi5, Rad51, or other recombination proteins

  • Investigate potential involvement in recombination-based processes similar to the Swi2-Swi5 complex

• Integration with genetic approaches:

  • Analyze protein behavior in strains defective in specific DNA replication or repair pathways

  • Correlate protein levels with phenotypic assays measuring genome stability

  • Investigate synthetic genetic interactions through protein analysis in double mutants

These investigations could provide insights into whether SPAC18B11.08c functions in DNA metabolism pathways similar to other characterized S. pombe proteins.

What are the considerations for developing a quantitative ELISA assay using the SPAC18B11.08c antibody?

Developing a quantitative ELISA for SPAC18B11.08c requires careful optimization of multiple parameters:

• Assay format selection:

  • Direct ELISA: Simplest approach, but may have lower sensitivity

  • Sandwich ELISA: Requires two antibodies recognizing different epitopes (may need additional antibody development)

  • Competitive ELISA: Useful for small proteins or specific epitope detection

• Optimization of critical parameters:

  • Coating conditions: Buffer composition, protein concentration, incubation time/temperature

  • Blocking protocol: Agent selection (BSA vs. casein), concentration, incubation time

  • Antibody concentration: Determine optimal working dilution through titration

  • Sample preparation: Lysis method, clarification steps, dilution series

  • Detection system: HRP vs. AP conjugates, substrate selection, development time

• Standard curve development:

  • If purified SPAC18B11.08c is available, create dilution series

  • If purified protein is unavailable, consider using standardized cell lysates

  • Include positive and negative control samples

  • Define limits of detection and quantification

• Validation requirements:

  • Precision: Intra-assay and inter-assay coefficients of variation

  • Accuracy: Recovery of spiked samples

  • Specificity: Cross-reactivity testing

  • Linearity: Dilution effects on measured concentrations

  • Stability: Effects of freeze-thaw cycles, storage conditions

A well-developed ELISA protocol would enable higher-throughput analysis of SPAC18B11.08c across multiple samples, facilitating larger-scale studies of protein regulation under various conditions.

How can the SPAC18B11.08c antibody contribute to broader studies of evolutionarily conserved proteins across yeast species?

The SPAC18B11.08c antibody can serve as a valuable tool for comparative evolutionary studies:

• Cross-species reactivity testing:

  • Evaluate antibody recognition of potential homologs in related yeast species

  • Test using Western blot on lysates from S. cerevisiae, C. albicans, and other fungi

  • Document epitope conservation through sequence alignment and structural prediction

  • Consider developing a panel of antibodies against conserved domains

• Functional conservation analysis:

  • Compare protein expression patterns across species under similar conditions

  • Analyze subcellular localization in different yeasts to infer conserved functions

  • Study protein-protein interactions across species to identify conserved complexes

  • Correlate with phenotypic data from orthologous gene mutations

• Integration with bioinformatics approaches:

  • Use antibody-generated data to validate computational predictions

  • Compare experimentally determined properties with those predicted from sequence

  • Build phylogenetic profiles based on antibody detection across species

  • Correlate protein conservation with functional domains

• Contribution to protein annotation:

  • Use antibody-derived data to improve annotation of uncharacterized proteins

  • Document post-translational modifications that may be conserved across species

  • Provide experimental evidence for predicted protein features

  • Support or challenge sequence-based orthology predictions

By extending studies beyond S. pombe, researchers can place SPAC18B11.08c in an evolutionary context, potentially revealing ancient conserved functions that might not be apparent from studies in a single species.