SPAC26H5.03 Antibody

What is SPAC26H5.03 protein and why is it significant in research?

SPAC26H5.03 (also known as pcf2) is an uncharacterized WD repeat-containing protein found in Schizosaccharomyces pombe (fission yeast). It functions as the CAF assembly factor (CAF-1) complex subunit B . This protein is particularly significant in research because:

• It belongs to the chromatin assembly factor complex, which plays a crucial role in nucleosome assembly during DNA replication and repair

• WD repeat-containing proteins typically serve as scaffolding platforms in multi-protein complexes

• Understanding its function contributes to our knowledge of chromatin regulation in eukaryotes

The protein has a molecular weight of approximately 57,426 Da and contains structural motifs that facilitate protein-protein interactions within larger complexes.

What experimental techniques can SPAC26H5.03 antibody be used for?

Based on available data, SPAC26H5.03 antibody has been validated for the following applications:

The antibody has been specifically validated with Schizosaccharomyces pombe (strain 972/24843) samples , making it particularly valuable for researchers studying protein complexes in fission yeast.

How can SPAC26H5.03 antibody be used to study protein complex assembly in fission yeast?

Studying protein complex assembly with SPAC26H5.03 antibody requires a methodological approach:

• Co-immunoprecipitation (Co-IP) protocols:

  • Lyse cells under non-denaturing conditions to preserve protein-protein interactions

  • Use SPAC26H5.03 antibody coupled to protein A/G beads to precipitate the target protein

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • Include appropriate controls (IgG control, lysate input)

• Chromatin immunoprecipitation (ChIP):

  • As a CAF-1 complex component, SPAC26H5.03 likely associates with chromatin

  • Cross-link proteins to DNA before immunoprecipitation

  • Use the antibody to pull down protein-DNA complexes

  • Analyze associated DNA sequences through sequencing

• Proximity ligation assays:

  • Combine SPAC26H5.03 antibody with antibodies against suspected interaction partners

  • Use species-specific secondary antibodies with oligonucleotide probes

  • Detect protein-protein interactions in situ through rolling circle amplification

The key insight from protein complex research is that assembly occurs in defined orders and stoichiometries. As noted in one study: "Assembly of protein complexes is likely to facilitate efficient assembly" .

What are the challenges in detecting low-abundance WD repeat proteins like SPAC26H5.03?

Detecting low-abundance WD repeat proteins presents several methodological challenges:

• Signal amplification strategies:

  • Use tyramide signal amplification (TSA) to enhance detection sensitivity

  • Employ biotin-streptavidin systems for multi-layer detection

  • Consider proximity ligation assays for in situ detection with single-molecule sensitivity

• Sample enrichment techniques:

  • Perform subcellular fractionation to concentrate the target protein

  • Use affinity purification targeting the WD repeat domain

  • Consider protein concentration techniques for dilute samples

• Background reduction methods:

  • Optimize blocking conditions (5% BSA or 5% milk in TBST)

  • Use highly specific secondary antibodies

  • Consider monovalent Fab fragments to reduce non-specific binding

Research indicates that "WD repeat proteins appear sensitive to aggregation when their binding partners are absent" , suggesting that maintaining native complex conditions during extraction may improve detection.

How should researchers optimize Western blot protocols for SPAC26H5.03 detection?

Optimizing Western blot protocols for SPAC26H5.03 requires methodical adjustment of several parameters:

• Sample preparation optimization:

  • Use extraction buffers containing protease inhibitors to prevent degradation

  • Consider adding phosphatase inhibitors if studying post-translational modifications

  • Determine optimal lysis conditions (detergent type and concentration)

• Blocking and antibody dilution optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Perform antibody titration (typically starting at 1:500 to 1:2000)

  • Optimize primary antibody incubation (overnight at 4°C vs. 1-2 hours at room temperature)

• Detection system selection:

  • Choose between chemiluminescence, fluorescence, or chromogenic detection

  • Consider the expected abundance of the target protein

  • Determine if multiplexing with other antibodies is required

For SPAC26H5.03 antibody specifically, available data suggests working dilutions in the range of 1:500 to 1:1000 for Western blot applications in fission yeast samples .

What controls should be included when using SPAC26H5.03 antibody in experimental workflows?

A rigorous experimental design with SPAC26H5.03 antibody requires comprehensive controls:

• Positive and negative sample controls:

  • Use wild-type S. pombe extracts as positive controls

  • Include SPAC26H5.03 deletion mutants as negative controls

  • Consider using the HDLM-2 and Daudi cell lines as reference samples (analogous to approaches used for other antibodies)

• Antibody validation controls:

  • Perform peptide competition assays to confirm specificity

  • Use isotype-matched control antibodies (rabbit IgG for the polyclonal antibody)

  • Include secondary antibody-only controls to assess background

• Loading and transfer controls:

  • Use housekeeping proteins (actin, tubulin) to normalize loading

  • Consider Ponceau S staining to confirm transfer efficiency

  • Include molecular weight markers to confirm target band size

• Experimental design controls:

  • Include biological replicates (minimum n=3)

  • Perform technical replicates for critical experiments

  • Consider using recombinant SPAC26H5.03 protein as a reference standard

What are common troubleshooting strategies when SPAC26H5.03 antibody yields inconsistent results?

When facing inconsistent results with SPAC26H5.03 antibody, consider these methodological approaches:

• Antibody storage and handling issues:

  • Check for antibody degradation (avoid repeated freeze-thaw cycles)

  • Ensure proper storage conditions (typically -20°C or -80°C)

  • Consider adding stabilizing proteins (BSA) to diluted antibody

• Sample preparation problems:

  • Verify complete protein denaturation for Western blot applications

  • Check for proteolytic degradation (add protease inhibitors)

  • Ensure complete cell lysis and protein solubilization

• Detection system optimization:

  • Adjust exposure times to avoid over/under-exposure

  • Try alternative secondary antibodies or detection methods

  • Consider using signal enhancers for weak signals

• Cross-reactivity investigation:

  • Perform immunoprecipitation followed by mass spectrometry to identify all bound proteins

  • Consider pre-adsorption with related proteins to improve specificity

  • Test the antibody on simpler samples (e.g., recombinant protein) to establish baseline performance

For fission yeast applications specifically, "small volumes of antibody vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage" , requiring brief centrifugation before use.

How can SPAC26H5.03 antibody be utilized in protein complex stoichiometry studies?

Analyzing protein complex stoichiometry with SPAC26H5.03 antibody requires sophisticated methodological approaches:

• Quantitative immunoprecipitation techniques:

  • Use calibrated recombinant protein standards for absolute quantification

  • Apply stable isotope labeling with amino acids in cell culture (SILAC) for relative quantification

  • Compare precipitated amounts of complex components using specific antibodies

• Native mass spectrometry integration:

  • Immunoprecipitate intact complexes under native conditions

  • Analyze complex composition and stoichiometry by native MS

  • Compare observed stoichiometries with predicted models

• Single-molecule approaches:

  • Use antibody-based fluorescent labeling for direct visualization

  • Apply step-wise photobleaching analysis to determine subunit counts

  • Correlate with biochemical data for comprehensive assessment

Research on protein complexes suggests that "maintenance of cellular stoichiometry" is crucial for proper function, making quantitative approaches essential for understanding SPAC26H5.03's role in the CAF-1 complex.

How does SPAC26H5.03 antibody compare to other tools for studying chromatin assembly factors?

When evaluating research tools for chromatin assembly factor studies, consider these methodological comparisons:

• Antibody-based vs. genetic approaches:

  Method	Advantages	Limitations
  SPAC26H5.03 antibody	Detects native protein, applicable to wild-type cells	May have cross-reactivity, limited to detection applications
  Tagged protein expression	Highly specific detection, customizable tags	May affect protein function, requires genetic modification
  CRISPR-Cas9 editing	Physiological expression levels, endogenous regulation	Technical complexity, potential off-target effects

• Cross-species considerations:

  • SPAC26H5.03 antibody is specifically validated for S. pombe

  • CAF-1 complex is evolutionarily conserved, but with species-specific variations

  • For mammalian studies, antibodies against p60 (human CAF-1 subunit B homolog) would be more appropriate

• Alternative detection methods:

  • Aptamer-based detection systems

  • Mass spectrometry-based proteomics

  • Functional complementation assays

Research on protein complexes indicates that "protein complex databases and repositories" can provide valuable comparative information for cross-species studies.

What emerging techniques might complement SPAC26H5.03 antibody-based research?

Future research with SPAC26H5.03 might benefit from these emerging methodological approaches:

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed spatial organization

  • Live-cell imaging with minimal antibody fragments

  • Correlative light and electron microscopy for structural context

• Novel proteomics approaches:

  • Proximity labeling (BioID, APEX) to identify interaction partners

  • Thermal proteome profiling to assess complex stability

  • Cross-linking mass spectrometry to map interaction interfaces

• Structural biology integration:

  • Cryo-electron microscopy of immunoprecipitated complexes

  • Integrative structural modeling using antibody epitope constraints

  • In-cell NMR to assess dynamics in native environments

• Single-cell applications:

  • Imaging mass cytometry with metal-conjugated antibodies

  • Single-cell Western blotting for heterogeneity assessment

  • Spatial transcriptomics correlation with protein localization

Recent developments in "computational prediction of protein complex structure" offer complementary approaches to antibody-based detection, allowing for integrated structural and functional studies.