SPCC16C4.04 Antibody

What is SPCC16C4.04 and what cellular functions might it be involved in?

SPCC16C4.04 belongs to a family of proteins found in Schizosaccharomyces pombe (fission yeast). Based on nomenclature and sequence analysis, it appears to be related to uncharacterized proteins that are unique components of the fission yeast Ino80 chromatin remodeling complex, similar to identified proteins such as SPCC1259.04, SPCC16C4.20, SPAC23G3.04, and SPAC144.02 .

The Ino80 complex in fission yeast is essential for cell viability and plays crucial roles in DNA damage response and replication stress management. Deletion of various Ino80 complex components (Arp8, Ies2, Ies6) results in sensitivity to hydroxyurea (HU), bleocin, and UV irradiation, indicating their involvement in DNA damage repair pathways . As a potential Ino80 complex-associated protein, SPCC16C4.04 might similarly participate in chromatin remodeling processes related to DNA repair and replication.

Methodologically, researchers should consider conducting genetic interaction studies through synthetic genetic arrays to identify functional relationships between SPCC16C4.04 and known DNA repair pathway components, similar to approaches used for other fission yeast proteins .

What are the recommended approaches for generating antibodies against fission yeast proteins like SPCC16C4.04?

For generating effective antibodies against fission yeast proteins:

• Select optimal immunization strategy: Target antigens directly to antigen-presenting cells to induce rapid and effective antibody responses .

• Utilize recombinant expression systems: Express the full-length SPCC16C4.04 protein or unique epitope regions in bacterial or mammalian expression systems.

• Consider both monoclonal and polyclonal approaches:

  • Monoclonal antibodies offer high specificity for single epitopes

  • Polyclonal antibodies recognize multiple epitopes, potentially increasing detection sensitivity

• Epitope selection: Analyze the SPCC16C4.04 sequence for unique, surface-exposed regions that differ from other Ino80 complex proteins to ensure specificity.

If investigating potential post-translational modifications (PTMs), implement additional purification steps - first deplete serum with unmodified target protein before performing affinity purification using the immobilized, modified target protein .

What validation techniques should be employed for antibodies against SPCC16C4.04?

A comprehensive validation strategy for SPCC16C4.04 antibodies should include:

• Western blot analysis:

  • Test against wild-type yeast extracts and SPCC16C4.04 deletion strains

  • Expected band size verification based on predicted molecular weight

  • Compare signals between FLAG/HA-tagged SPCC16C4.04 strains and untagged controls

• Immunoprecipitation validation:

  • Perform IP followed by mass spectrometry to confirm target specificity

  • Verify antibody performance using epitope-tagged constructs as controls

• Cross-reactivity assessment:

  • Test against closely related proteins like SPCC16C4.20 to ensure specificity

  • Evaluate performance in various buffer conditions (varying salt concentrations)

• For phospho-specific antibodies:

  • Treatment with appropriate kinase activators/inhibitors

  • Phosphatase treatment to verify phosphospecificity

  • Comparison with point mutation controls

  • Peptide competition assays with phosphorylated vs. non-phosphorylated peptides

• ChIP validation (if applicable):

  • Include negative control antibodies and control primers

  • Perform ChIP-qPCR at known genomic loci to establish enrichment patterns

What are the optimal lysis and extraction conditions for detecting SPCC16C4.04 in fission yeast?

Based on protocols used for Ino80 complex proteins in fission yeast:

• Cell preparation:

  • Grow cultures to optimal density (2 × 10⁸ cells/ml)

  • Harvest and wash cells in ice-cold PBS

• Lysis buffer composition:

  • Base buffer: H buffer (HEPES-KCl) with salt concentrations between 300-500 mM KCl

  • Add Complete EDTA-free protease inhibitor cocktail (Roche)

  • Include 0.1% SDS, 1% Triton X, 0.1% sodium deoxycholate, 1 mM EDTA

• Cell disruption method:

  • Freeze cells in liquid nitrogen

  • Break in dry ice using mechanical disruption (coffee mill method)

  • Alternative: use FastPrep machine (MPBio) for cell wall disruption

• Extract clarification:

  • Centrifuge lysate at high speed (37K in a SW40 Beckman rotor)

  • Carefully collect supernatant containing soluble proteins

• Protein quantification:

  • Determine protein concentration using SDS-PAGE with Coomassie blue staining

  • Compare band intensity with bovine serum albumin standards

How can I assess the specificity of my SPCC16C4.04 antibody in different experimental applications?

To thoroughly assess antibody specificity across applications:

For antibodies targeting modified forms, include phosphatase treatment (for phospho-antibodies) or other modification-removing enzymes to verify signal specificity to the targeted modification .

What are the methodological considerations for using SPCC16C4.04 antibodies in chromatin immunoprecipitation (ChIP) experiments?

When designing ChIP experiments with SPCC16C4.04 antibodies:

• Crosslinking protocol optimization:

  • Standard formaldehyde crosslinking (1% final concentration for 1 hour at room temperature)

  • Quench with glycine (0.125 M final concentration)

• Chromatin fragmentation:

  • Sonicate to achieve fragment sizes of 500-1000 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Immunoprecipitation conditions:

  • Protein A-Sepharose beads for capture

  • Include appropriate controls:

    • Input chromatin (pre-IP sample)

    • No-antibody control

    • Non-specific IgG control

• Washing conditions:

  • Wash beads 5 times with wash buffer (0.1% SDS, 50 mM HEPES-KOH pH 7.5, 1% Triton X, 0.1% sodium deoxycholate, 1 mM EDTA, 500 mM NaCl)

  • Followed by washes with lower salt buffer (150 mM KCl)

• Signal quantification:

  • Use SYBR green incorporation

  • Quantify using real-time PCR systems (e.g., ABI Prism 7700)

  • Calculate percent chromatin immunoprecipitated from input

  • Include both positive and negative genomic control regions

• ChIP validation approach:

  • Consider using the ChIPAb+ approach - validate with defined primer sets for known genomic loci

  • Include negative control antibodies and appropriate control primers

How can I investigate potential interactions between SPCC16C4.04 and the Ino80 complex?

To characterize protein-protein interactions within chromatin remodeling complexes:

• Co-immunoprecipitation strategy:

  • Generate epitope-tagged strains (FLAG, HA, or MYC tags)

  • Perform reciprocal co-IP experiments (pull down SPCC16C4.04 and probe for Ino80 components, and vice versa)

  • Elute using specific methods:

    • FLAG-tagged proteins: elute with FLAG peptide (2.5 mg/ml)

    • HA-tagged proteins: standard elution methods

    • MYC-tagged proteins: elute with SDS loading buffer

• Mass spectrometry identification:

  • Process immunoprecipitated samples for LC-MS/MS analysis:

    • Reduce and carbamidomethylate samples

    • Digest with trypsin (10 ng/μl modified trypsin in 25 mM ammonium bicarbonate with 30% acetonitrile)

    • Analyze peptides using liquid chromatography coupled to MS/MS

• Data analysis approach:

  • Search against eukaryote entries in protein databases (e.g., Uniprot)

  • Use analysis software such as Mascot (Matrix Science)

  • Compare identified interacting proteins with known Ino80 components

• Confirming direct interactions:

  • Consider yeast two-hybrid assays

  • In vitro binding assays with recombinant proteins

  • Proximity-based labeling methods (BioID, APEX)

What technical considerations should be made when investigating SPCC16C4.04's potential role in DNA damage response pathways?

To elucidate functional roles in DNA damage response:

• Genetic interaction analysis:

  • Create deletion mutants and analyze epistatic relationships

  • Test sensitivity to DNA-damaging agents:

    • Hydroxyurea (HU) for replication stress

    • Bleocin for single and double-strand breaks

    • UV irradiation for pyrimidine dimers

  • Combine with deletions in known DNA repair genes to identify pathways:

    • Fan1 and Pso2 pathways for interstrand crosslink repair

    • Rhp18 (post-replication repair)

    • Rhp51 (homologous recombination)

• Viability assay protocol:

  • Grow cells overnight at 30°C

  • Prepare serial dilutions (typically five 10-fold dilutions)

  • Spot onto agar plates with/without DNA damaging agents

  • Analyze growth after 3-5 days

• Microscopy approaches:

  • DAPI staining to assess nuclear morphology

  • Monitor SPCC16C4.04 localization after DNA damage

  • Quantify cell cycle progression or arrest following damage

• Chromatin association dynamics:

  • Use ChIP to monitor recruitment to damaged DNA

  • Analyze temporal dynamics of chromatin association

  • Identify specific genomic regions where SPCC16C4.04 may function

How can I develop antibodies specific to post-translationally modified forms of SPCC16C4.04?

For generating modification-specific antibodies:

• Modification site identification:

  • Perform mass spectrometry analysis to identify potential PTM sites

  • Focus on evolutionarily conserved residues

  • Consider known modification patterns of related proteins

• Immunization strategy for PTM-specific antibodies:

  • Use synthetic peptides containing the specific modification

  • Design peptides with the modification centrally positioned

  • Consider coupling to carrier proteins to enhance immunogenicity

• Purification approach for maximal specificity:

  • Initial depletion using unmodified protein/peptide affinity column

  • Followed by positive selection using modified protein/peptide column

  • This two-step approach significantly enhances specificity for the modified form

• Validation protocols specific for PTM antibodies:

  • Treatment with appropriate enzymes to remove modifications:

    • Phosphatases for phospho-specific antibodies

    • Deubiquitinating enzymes for ubiquitin-specific antibodies

  • Compare recognition in cells with/without point mutations at modification sites

  • Peptide competition assays with modified and unmodified peptides

• Quantitative assessment of cross-reactivity:

  • Peptide arrays containing modified and unmodified variants

  • ELISA-based testing of antibody specificity

  • Western blotting under varying conditions to assess robustness

What approaches can resolve contradictory results when using different SPCC16C4.04 antibodies in experimental settings?

When faced with conflicting antibody results:

• Comprehensive epitope mapping:

  • Determine the exact binding regions of each antibody

  • Assess whether antibodies recognize different domains that may behave differently under experimental conditions

• Comparative validation using multiple techniques:

  • Cross-validate using orthogonal methods:

    • Western blot

    • Immunoprecipitation

    • ChIP

    • Mass spectrometry

• Strain verification and controls:

  • Generate epitope-tagged constructs as positive controls

  • Create deletion strains as negative controls

  • Use purified recombinant protein as standard

• Buffer and protocol optimization matrix:

  • Systematically test different:

    • Salt concentrations (150-500 mM KCl/NaCl)

    • Detergent combinations (SDS, Triton X, sodium deoxycholate)

    • Fixation conditions

    • Incubation times and temperatures

• Independent validation by mass spectrometry:

  • Analyze immunoprecipitated material by LC-MS/MS

  • Compare peptide coverage and protein identification confidence scores

  • Use isotope labeling strategies for quantitative comparisons

• Technical verification table: