SPAC27E2.14 Antibody

Basic Research Questions

• What is SPAC27E2.14 and why would researchers need an antibody against it?

  SPAC27E2.14 is classified as a sequence orphan or hypothetical protein in Schizosaccharomyces pombe (strain 972 / ATCC 24843) . As an uncharacterized protein with no known homologs, antibodies against it are crucial for validating its expression, determining subcellular localization, and characterizing its interactions with other cellular components. The computed structure model (AF_AFC6Y4B0F1) indicates a moderate confidence level (pLDDT global score of 64.45), suggesting a partially structured protein .

• What types of SPAC27E2.14 antibodies are currently available for research?

  Currently, polyclonal antibodies raised against recombinant Schizosaccharomyces pombe SPAC27E2.14 protein are available for research purposes. These antibodies are typically:

  • Raised in rabbits

  • Antigen-affinity purified

  • Supplied in liquid form (typically in 50% glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative)

  • Validated for applications such as ELISA and Western blotting

  These antibodies are designed specifically for research use and not for diagnostic or therapeutic applications.

• What experimental applications are SPAC27E2.14 antibodies validated for?

  According to product documentation, current SPAC27E2.14 antibodies have been tested and validated for:

  • ELISA (Enzyme-Linked Immunosorbent Assay)

  • Western blotting (WB) for identification of the antigen

  Researchers should validate these antibodies for other applications such as immunoprecipitation, immunofluorescence microscopy, or ChIP (Chromatin Immunoprecipitation) before proceeding with experiments.

• How should SPAC27E2.14 antibodies be stored and handled in laboratory settings?

  For optimal performance and longevity:

  • Store at -20°C or -80°C upon receipt

  • Avoid repeated freeze-thaw cycles

  • When working with the antibody, keep it on ice

  • For long-term storage of aliquots, use -80°C

  • Before use, centrifuge the antibody vial briefly to collect all material at the bottom

• What controls should be included when using SPAC27E2.14 antibodies?

  When designing experiments with SPAC27E2.14 antibodies, include:

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

  • Negative control: Lysate from a SPAC27E2.14 deletion strain (if available)

  • Isotype control: Non-specific IgG from the same species the antibody was raised in (rabbit)

  • Loading control: Antibody against a constitutively expressed S. pombe protein

  • Pre-adsorption control: SPAC27E2.14 antibody pre-incubated with excess recombinant protein

Advanced Research Questions

• How can researchers validate the specificity of SPAC27E2.14 antibodies given the limited information about this uncharacterized protein?

  For rigorous validation of antibody specificity:

  1. Generate a SPAC27E2.14 knockout strain using CRISPR-Cas9 or traditional homologous recombination methods

  2. Create an epitope-tagged version (e.g., GFP, FLAG, or HA tag) of SPAC27E2.14 expressed under its native promoter

  3. Perform parallel Western blots with both the SPAC27E2.14 antibody and an anti-tag antibody

  4. Conduct immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein

  5. Perform immunodepletion experiments with recombinant SPAC27E2.14 protein

  These approaches collectively provide strong evidence for antibody specificity to the target protein.

• What methodological considerations should be taken into account when using SPAC27E2.14 antibodies for immunofluorescence in S. pombe?

  For successful immunofluorescence in fission yeast:

  1. Cell wall digestion: Optimize spheroplasting using zymolyase or lysing enzymes to ensure adequate antibody penetration while maintaining cellular morphology

  2. Fixation method: Compare methanol fixation and paraformaldehyde fixation, as described in search result for S. pombe immunofluorescence

  3. Blocking: Use 5% BSA or normal serum from the secondary antibody species to reduce background

  4. Antibody dilution: Perform titration experiments (starting from manufacturer's recommendation of ≤5 μg/mL)

  5. Microscopy controls: Include secondary-only controls and cells without the target protein

  6. Counterstaining: Use DAPI for nuclear staining to aid in cellular localization analysis

• How can SPAC27E2.14 antibodies be used to investigate protein-protein interactions and complex formation?

  To investigate protein-protein interactions:

  1. Co-immunoprecipitation (Co-IP):

     • Optimize lysis conditions to preserve native protein complexes

     • Use crosslinking agents like formaldehyde or DSP to stabilize transient interactions

     • Elute with gentle conditions to maintain complex integrity

  2. Proximity Ligation Assay (PLA):

     • Combine SPAC27E2.14 antibody with antibodies against suspected interaction partners

     • Use species-specific PLA probes and optimize signal amplification

  3. Immunoprecipitation coupled to mass spectrometry (IP-MS):

     • Perform stringent washing steps to remove non-specific binders

     • Include appropriate negative controls (IgG, knockout cells)

     • Use both label-free and SILAC approaches for quantitative comparisons

  4. Size Exclusion Chromatography followed by Western blot:

     • Analyze whether SPAC27E2.14 co-elutes with suspected complex components

• What approaches should be used to characterize post-translational modifications of SPAC27E2.14 using available antibodies?

  For PTM characterization:

  1. Phosphorylation analysis:

     • Treat samples with phosphatase inhibitors during preparation

     • Perform Phos-tag SDS-PAGE to separate phosphorylated forms

     • Compare migration patterns before and after phosphatase treatment

  2. Glycosylation analysis:

     • Based on S. pombe glycosylation patterns described in search result , treat samples with endoglycosidases (EndoH)

     • Compare migration patterns to identify glycosylated forms

  3. Ubiquitination analysis:

     • Use denaturing conditions during immunoprecipitation

     • Probe with anti-ubiquitin antibodies after SPAC27E2.14 immunoprecipitation

  4. Mass spectrometry:

     • Immunoprecipitate SPAC27E2.14 and analyze by LC-MS/MS

     • Use enrichment strategies specific for phosphopeptides, glycopeptides, etc.

• How can SPAC27E2.14 antibodies be used in combination with genomic and transcriptomic data to understand protein function?

  For integrative analysis:

  1. Correlation with transcriptome data:

     • Use tiling array and Illumina sequencing profiles available for SPAC27E2.14

     • Compare protein expression (Western blot) with mRNA levels across conditions

  2. ChIP-seq analysis (if nuclear protein):

     • Optimize cross-linking conditions for S. pombe

     • Use stringent controls for antibody specificity

     • Compare binding sites with transcriptional changes

  3. Proteomics correlation:

     • Combine antibody-based quantification with global proteomics data

     • Identify co-regulated proteins across conditions

  4. Genetic interaction mapping:

     • Use antibody to validate protein levels in genetic interaction screens

     • Confirm protein expression changes in synthetic lethal or suppressor strains

• What methodological approaches should be considered when using SPAC27E2.14 antibodies for analyzing protein localization during cell cycle progression in S. pombe?

  For cell cycle localization studies:

  1. Synchronization methods:

     • Optimize lactose gradient, nitrogen starvation, or cdc25-22 temperature-sensitive mutant approaches

     • Confirm synchronization by flow cytometry (FACS) with DAPI staining

  2. Time-course sampling:

     • Collect samples at defined intervals covering the entire cell cycle

     • Process samples simultaneously for immunofluorescence

  3. Co-localization with markers:

     • Use antibodies against known cell cycle-regulated proteins

     • Include markers for cellular structures (SPB, septum, etc.)

  4. Quantitative analysis:

     • Measure signal intensity relative to cell cycle progression

     • Correlate with septum formation and nuclear division

  5. Live-cell imaging comparison:

     • Compare immunofluorescence results with live-cell imaging of tagged protein

• How can researchers troubleshoot non-specific binding when using SPAC27E2.14 antibodies in S. pombe lysates?

  To address non-specific binding:

  1. Optimization of blocking conditions:

     • Test different blocking agents (BSA, milk, normal serum)

     • Increase blocking time and concentration

  2. Antibody purification strategies:

     • Consider pre-adsorption against fixed S. pombe SPAC27E2.14 deletion strain

     • Use antigen-specific affinity purification to enrich specific antibodies

  3. Sample preparation optimization:

     • Test different lysis methods (mechanical disruption, enzymatic)

     • Optimize detergent concentrations to reduce non-specific interactions

  4. Western blot conditions:

     • Increase washing stringency (higher salt, mild detergents)

     • Optimize antibody dilution and incubation conditions

     • Consider using more sensitive detection methods with shorter exposure times

Table 1: SPAC27E2.14 Antibody Properties and Applications

Table 2: SPAC27E2.14 Protein Characteristics

Table 3: Experimental Design for SPAC27E2.14 Antibody Validation