SPCC1235.01 Antibody

What is SPCC1235.01 and why is it significant in fission yeast research?

SPCC1235.01 is a protein coding gene in Schizosaccharomyces pombe (fission yeast). Antibodies against this protein are valuable tools for studying protein expression, localization, and function in this model organism. Fission yeast serves as an excellent eukaryotic model system due to its relatively simple genome and conserved cellular processes that parallel those in higher eukaryotes, including humans. The study of SPCC1235.01 contributes to our understanding of fundamental cellular processes in eukaryotic cells .

What experimental applications are most suitable for SPCC1235.01 antibodies?

Based on similar S. pombe antibodies, SPCC1235.01 antibodies are typically applicable for multiple experimental techniques including Western blotting (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) . For comprehensive characterization studies, researchers should consider additional applications such as immunoprecipitation (IP), immunofluorescence (IF), and chromatin immunoprecipitation (ChIP) depending on the specific research question and antibody properties.

How do I validate the specificity of a SPCC1235.01 antibody?

Antibody validation requires multiple approaches:

• Positive control: Use recombinant SPCC1235.01 protein (approximately 200μg) as provided in antibody kits

• Negative control: Pre-immune serum should show no reactivity with the target protein

• Genetic validation: Test the antibody in SPCC1235.01 deletion strains

• Molecular weight confirmation: Verify that detected bands match the predicted molecular weight

• Cross-reactivity assessment: Test against related S. pombe proteins to confirm specificity

How should I design experiments to study protein-protein interactions involving SPCC1235.01?

When designing experiments to investigate protein-protein interactions:

• Select appropriate epitope tags that don't interfere with protein function

• Consider both co-immunoprecipitation using SPCC1235.01 antibodies and reciprocal pull-downs

• Include proper negative controls (pre-immune serum)

• Confirm interactions using multiple methodologies (yeast two-hybrid, proximity labeling)

• Validate physiological relevance through genetic studies

• Consider differential extraction methods to account for membrane association or insoluble fractions

What are the optimal storage and handling conditions for SPCC1235.01 antibodies?

For maximum stability and performance:

• Store antibodies at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• For working stocks, store at 4°C with preservatives such as sodium azide (0.02%)

• Prior to use, centrifuge antibody solutions briefly to collect contents at the bottom of the vial

• Handle according to the isotype specifications (most commonly IgG)

• Monitor antibody performance over time with consistent positive controls

How can I optimize immunoprecipitation protocols for SPCC1235.01 studies in fission yeast?

Optimizing immunoprecipitation for SPCC1235.01 requires:

• Cell lysis considerations:

  • Use gentle detergents (0.5-1% NP-40 or Triton X-100) for membrane proteins

  • Include protease and phosphatase inhibitors

  • Optimize salt concentration (150-300mM NaCl) to maintain interactions while reducing background

• Antibody coupling:

  • Pre-clear lysates with protein A/G beads

  • Use optimized antibody:lysate ratios (typically 2-5μg antibody per 500μg-1mg protein)

  • Consider cross-linking antibodies to beads to prevent co-elution

• Controls:

  • Include pre-immune serum as negative control

  • Use untagged strains as additional controls

  • Perform reciprocal IPs when studying interactions

What strategies can overcome challenges in detecting low-abundance SPCC1235.01 protein?

For detecting low-abundance proteins:

• Enrichment strategies:

  • Subcellular fractionation to concentrate relevant cellular compartments

  • Affinity purification using tagged constructs

  • Immunoprecipitation followed by Western blotting

• Signal amplification:

  • Use high-sensitivity detection systems (ECL Prime or Femto)

  • Consider tyramide signal amplification for immunofluorescence

  • Explore proximity ligation assays for interaction studies

• Expression optimization:

  • Use promoter systems that provide moderate overexpression

  • Consider synchronizing cells if protein levels vary during cell cycle

  • Use proteasome inhibitors if protein is rapidly degraded

How do I interpret contradictory results between different antibody-based detection methods for SPCC1235.01?

When facing contradictory results:

• Review antibody characteristics:

  • Epitope locations may affect accessibility in different techniques

  • Polyclonal antibodies recognize multiple epitopes, providing different sensitivity than monoclonals

  • Consider if post-translational modifications affect epitope recognition

• Methodological factors:

  • Protein denaturation in Western blots versus native conditions in IF

  • Fixation methods may alter epitope accessibility

  • Buffer conditions may affect antibody binding

• Validation approaches:

  • Use multiple antibodies targeting different epitopes

  • Complement with non-antibody methods (MS/MS, activity assays)

  • Tag the protein at different positions (N vs C-terminal)

  • Consider genetic approaches (deletion, point mutations)

How can I distinguish between specific and non-specific signals in SPCC1235.01 antibody experiments?

To distinguish specific from non-specific signals:

• Essential controls:

  • Pre-immune serum negative control

  • SPCC1235.01 deletion strains

  • Peptide competition assays

  • Isotype-matched control antibodies

• Signal characteristics:

  • Evaluate signal intensity relative to background

  • Assess signal consistency across replicates

  • Compare with predicted molecular weight and localization patterns

  • Evaluate cross-reactivity with related proteins

• Multiple detection methods:

  • Confirm key findings with orthogonal techniques

  • Use tagged versions of the protein as reference points

  • Consider mass spectrometry validation of detected bands

What modifications to standard protocols are needed when using SPCC1235.01 antibodies in different S. pombe strain backgrounds?

When adapting protocols for different strain backgrounds:

• Genetic considerations:

  • Verify protein sequence conservation across laboratory strains

  • Account for strain-specific post-translational modifications

  • Consider genetic interactions that might affect protein expression

• Procedural adjustments:

  • Optimize cell lysis conditions for different cell wall properties

  • Adjust antibody concentrations based on expression levels

  • Modify incubation times for strains with different growth characteristics

• Validation strategies:

  • Include wild-type strain 972 (ATCC 24843) as a reference

  • Compare results across multiple strain backgrounds

  • Document strain-specific variations in antibody performance

How can I combine SPCC1235.01 antibody studies with genetic approaches to enhance research outcomes?

Integrating antibody studies with genetic approaches:

• Complementary techniques:

  • Use CRISPR/Cas9 to create tagged versions or knockout strains

  • Employ temperature-sensitive mutants for conditional studies

  • Implement auxin-inducible degron systems for rapid protein depletion

• Experimental design:

  • Start with genetic characterization followed by biochemical validation

  • Use suppressor screens to identify functional interactors

  • Implement synthetic genetic array analysis to map genetic networks

• Data integration:

  • Correlate antibody-detected protein levels with phenotypic outcomes

  • Combine localization studies with functional genetic assays

  • Use quantitative approaches to relate protein abundance to function

What are common pitfalls when using SPCC1235.01 antibodies in fission yeast research and how can they be addressed?

Common challenges and solutions:

• High background in Western blots:

  • Increase blocking stringency (5% BSA or milk)

  • Optimize antibody dilutions (typically start with 1:1000)

  • Increase wash duration and number of washes

  • Consider alternative membrane types (PVDF vs nitrocellulose)

• Inconsistent immunoprecipitation results:

  • Optimize lysis conditions to preserve protein interactions

  • Ensure antibody quality hasn't degraded over time

  • Consider crosslinking prior to cell lysis for transient interactions

  • Add competing peptides to reduce non-specific binding

• Poor signal in immunofluorescence:

  • Test multiple fixation methods (formaldehyde, methanol)

  • Optimize permeabilization conditions

  • Implement antigen retrieval techniques

  • Consider signal amplification methods

How do post-translational modifications affect SPCC1235.01 antibody recognition and experimental outcomes?

Post-translational modifications can significantly impact antibody recognition:

• Common modifications in yeast proteins:

  • N-glycosylation at asparagine residues in sequons (N-X-S/T)

  • Phosphorylation at serine, threonine, and tyrosine residues

  • Ubiquitination at lysine residues

• Experimental considerations:

  • Use phospho-specific antibodies for phosphorylation studies

  • Employ enzymatic treatments (phosphatase, glycosidase) to reveal masked epitopes

  • Consider modification-specific antibodies for specialized studies

  • Use mass spectrometry to map modification sites

• Interpretation guidelines:

  • Multiple bands may indicate modified forms rather than degradation

  • Shifts in apparent molecular weight may reflect modifications

  • Temporal changes may indicate dynamic regulation via modifications