SPAC3H5.11 Antibody

Basic Research Questions

• What is SPAC3H5.11 and what are the key considerations for antibody selection?

SPAC3H5.11 is an uncharacterized kinase in Schizosaccharomyces pombe (fission yeast). When selecting an antibody for this protein, researchers should consider:

• Specificity for SPAC3H5.11 without cross-reactivity to other S. pombe proteins

• Recognition of native versus denatured forms of the protein

• Application compatibility (Western blotting, immunoprecipitation, etc.)

• Validated performance in S. pombe lysates

• Species of origin to avoid interference in co-immunoprecipitation experiments

Custom antibodies against SPAC3H5.11 are available from specialty suppliers with sizes typically offered in 0.1ml or 2ml formats .

• What validation methods should be used to confirm SPAC3H5.11 antibody specificity?

Thorough validation is critical for antibodies against uncharacterized proteins. Recommended approaches include:

• Testing against wild-type and SPAC3H5.11 deletion strains

• Western blot analysis to confirm expected molecular weight (comparing to predicted size from sequence data)

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Testing for cross-reactivity against proteome arrays containing S. pombe proteins

Research has shown that even highly specific antibodies can unexpectedly cross-react with noncognate proteins, making comprehensive validation essential. One study screening 11 antibodies against approximately 5,000 yeast proteins found varying degrees of cross-reactivity that could not be predicted by sequence alignment alone .

• What sample preparation methods yield optimal results with SPAC3H5.11 antibody?

For S. pombe proteins like SPAC3H5.11, optimal sample preparation includes:

For immunoprecipitation experiments, researchers should follow protocols similar to those used for other S. pombe proteins: "After analyzing the expression of protein in co-transformed cells by Western blotting using anti-His or anti-FLAG antibody, the anti-FLAG M2 affinity gel can be used to immunoprecipitate FLAG-fused protein" .

• What are the common applications of SPAC3H5.11 antibody in fission yeast research?

SPAC3H5.11 antibody supports multiple research applications:

• Western blotting to detect expression levels and modifications

• Immunoprecipitation to identify interaction partners

• Chromatin immunoprecipitation (ChIP) if SPAC3H5.11 has nuclear functions

• Immunofluorescence microscopy to determine subcellular localization

• Flow cytometry for cell population analysis

Depending on research goals, the antibody can be used similarly to those for characterized kinases in S. pombe to study regulatory pathways, stress responses, or cell cycle control.

Advanced Research Questions

• How can researchers apply high-throughput screening methods with SPAC3H5.11 antibody?

Advanced screening approaches include:

High-throughput methodologies can be adapted from successful antibody screening studies. For example, researchers have identified specific antibodies against target proteins using "high-throughput single-cell RNA and VDJ sequencing" techniques . For SPAC3H5.11, similar approaches could involve:

• Screening SPAC3H5.11 interactions using antibody arrays or protein microarrays

• Implementing single-cell approaches to study kinase activation patterns

• Developing multiplex assays to simultaneously monitor multiple signaling pathways

A comprehensive experimental design would include:

• Initial expression profiling across different growth conditions

• Identification of stimuli that alter SPAC3H5.11 expression or modification

• Large-scale co-immunoprecipitation followed by mass spectrometry

• Validation of key interactions with reciprocal immunoprecipitation

• What approaches can identify SPAC3H5.11 kinase substrates using antibody-based techniques?

Identifying kinase substrates requires specialized methodologies:

• Antibody-based substrate trapping: Using SPAC3H5.11 antibody to co-immunoprecipitate substrate complexes after crosslinking

• Phospho-specific antibody screening: Developing antibodies against predicted phosphorylation motifs of SPAC3H5.11

• Chemical genetics approach: Combining SPAC3H5.11 antibody with analog-sensitive kinase mutants

The implementation could follow established protocols: "For chemical cross-linking, endogenous proteins can be metabolically labeled with [35S]Met/Cys, and target proteins immunopurified with a covalently immobilized monoclonal antibody. The immunopurified proteins can then be exposed in vitro to reversible cross-linkers like DSP" .

• How can researchers address epitope masking when SPAC3H5.11 forms protein complexes?

Epitope masking occurs when protein-protein interactions block antibody recognition sites. Strategies to overcome this include:

• Using multiple antibodies targeting different regions of SPAC3H5.11

• Employing native versus denaturing conditions strategically

• Implementing epitope retrieval techniques for fixed samples

• Utilizing proximity labeling techniques (BioID, APEX) as complementary approaches

For optimal epitope prediction, researchers can employ "Alphafold2 and molecular docking methods" similar to those used to identify antigenic epitopes in other systems .

• What techniques can be used to monitor SPAC3H5.11 dynamics during the cell cycle?

To study SPAC3H5.11 throughout the cell cycle:

• Synchronize S. pombe cultures using standard methods (nitrogen starvation, hydroxyurea block, or cdc25-22 temperature-sensitive mutants)

• Collect time-point samples for Western blot analysis with SPAC3H5.11 antibody

• Combine with flow cytometry using DNA content markers

• Employ real-time imaging with fluorescently tagged antibodies in permeable cells

Quantification methods should follow established protocols: "Real-time quantitative PCR can be performed with 4 μg of total RNA prepared from fission yeast using TRIzol and reverse transcribed to first strand cDNA. PCR amplification reactions can be performed using SYBR Premix on an ABI Prism sequence detection system" .

• How do different expression systems affect the quality of recombinant SPAC3H5.11 for antibody production?

The expression system significantly impacts protein quality for antibody production:

These options are available for recombinant SPAC3H5.11 production , allowing researchers to select the system that best matches their antibody requirements.

• How can researchers implement ChIP-seq with SPAC3H5.11 antibody to study potential DNA interactions?

If SPAC3H5.11 has DNA-binding properties, ChIP-seq can be implemented following these guidelines:

• Chromatin preparation: Crosslink S. pombe cells with formaldehyde (typically 1%) for 15-30 minutes

• Sonication: Fragment chromatin to 200-500bp pieces

• Immunoprecipitation: Using SPAC3H5.11 antibody with appropriate controls

• Library preparation and sequencing: Following standard NGS protocols

• Data analysis: Align to S. pombe genome and identify enriched regions

Researchers can adapt established protocols: "For ChIP assays, yeast cells can be incubated separately overnight with 20 μl of protein A/G-Sepharose and 10 μl of the appropriate antibody. DNA recovered from the immunoprecipitated fractions can be amplified using PCR with specific primers" .

• What strategies can resolve contradictory results between different lots of SPAC3H5.11 antibody?

When facing inconsistent results between antibody lots:

• Implement systematic validation for each lot using positive and negative controls

• Compare epitope recognition through peptide competition assays

• Evaluate batch-to-batch variability through side-by-side Western blots

• Consider monoclonal alternatives if polyclonal antibodies show high variability

• Maintain detailed records of antibody performance across experiments

Research has demonstrated that even well-characterized antibodies can display significant variability between lots, necessitating rigorous quality control measures for each new batch .

• How can genetic approaches complement SPAC3H5.11 antibody studies?

Integrating genetic approaches enhances antibody-based research:

• Create tagged SPAC3H5.11 strains (GFP, FLAG, HA) for parallel validation

• Generate temperature-sensitive or analog-sensitive SPAC3H5.11 mutants

• Implement CRISPR-based approaches for precise genome editing

• Develop conditional expression systems to control SPAC3H5.11 levels

Tagged versions can be particularly valuable: "ΔPng1 cells co-transformed with pJR2–41U-Png1-His 6 and pREP1–3×FLAG-Mst1 and cultured in the EMM medium without uracil and leucine overnight" represent an example of this complementary approach in S. pombe .