SPBC1347.09 Antibody

What is SPBC1347.09 in Schizosaccharomyces pombe, and why is it studied?

SPBC1347.09 is a protein-coding gene in the fission yeast Schizosaccharomyces pombe (strain 972 / ATCC 24843). While the specific function of this protein remains under investigation, it is part of a growing body of research into the functional characterization of the fission yeast proteome. Fission yeast serves as an excellent model organism for studying fundamental cellular processes due to its relatively small genome (approximately 5,100 genes), making it valuable for system-wide studies of gene and protein networks .

Methodological approach: To study this protein, researchers typically employ:

• Gene deletion techniques to create SPBC1347.09Δ strains

• Epitope tagging (often with HA or FLAG tags) for protein detection

• Expression analysis under various conditions using RNA-Seq

• Protein localization studies using fluorescence microscopy

How can I confirm the specificity of a commercial SPBC1347.09 antibody?

Confirming antibody specificity is crucial for reliable results in S. pombe research:

Recommended validation approaches:

The commercially available antibodies (e.g., CSB-PA528481XA01SXV) are typically antigen-affinity purified polyclonal antibodies raised in rabbits against recombinant SPBC1347.09 protein, providing good specificity .

What protocols are most effective for Western blot detection of SPBC1347.09?

For optimal Western blot detection of SPBC1347.09 in fission yeast:

Sample preparation:

• Collect cells by centrifugation from mid-log phase cultures

• Lyse cells using glass bead method in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NonidetP-40, 0.1% SDS, 12 mM sodium deoxycholate)

• Centrifuge at 12,000 rpm for 15 min at 4°C to remove debris

• Resolve proteins by SDS-PAGE on 12% Bis-Tris gel

• Transfer onto PVDF membrane

Detection recommendations:

• Primary antibody: Use SPBC1347.09 antibody at 1:10,000 dilution

• Secondary antibody: HRP-conjugated anti-rabbit at 1:100,000 dilution

• Detection: ECL chemiluminescence system

• Controls: Include SPBC1347.09Δ strain as negative control and tubulin (TAT-1 antibody) as loading control

How can I optimize co-immunoprecipitation studies with SPBC1347.09?

Co-immunoprecipitation (Co-IP) is valuable for understanding protein-protein interactions involving SPBC1347.09:

Optimization protocol:

• Create strains with epitope-tagged SPBC1347.09 (e.g., 3×HA tag) integrated at the chromosomal locus

• Harvest cells from appropriate conditions (consider testing multiple stress conditions)

• Prepare cell lysates as described for Western blotting

• Incubate with 5 μg anti-HA antibody overnight at 4°C

• Add Protein A/G magnetic beads and incubate at 4°C for 4 hours

• Wash beads 6× with buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% NonidetP-40, 5% glycerol)

• Elute immunoprecipitates by boiling in SDS-PAGE loading buffer

• Analyze by Western blotting with appropriate antibodies

Critical considerations:

• Salt concentration in washing buffer affects stringency; optimize to reduce background while maintaining true interactions

• Consider crosslinking before lysis for transient interactions

• Validate interactions with reciprocal Co-IPs using differently tagged proteins

How does SPBC1347.09 expression change under stress conditions?

While specific data on SPBC1347.09 expression changes isn't provided in the search results, fission yeast stress response studies typically follow this methodology:

Standard approach for expression analysis:

• Expose cells to specific stressors (e.g., diamide for disulfide stress, nutrient limitation, heat)

• Collect samples at specific time intervals (e.g., 0, 20, 60, 90, 120 min)

• Extract total RNA using standard methods (e.g., glass bead method with ISOGEN reagent)

• Perform RNA-Seq or qRT-PCR analysis

• Normalize expression to housekeeping genes like act1+

For qRT-PCR:

• Use cDNA synthesis (e.g., ReverTra Ace-α-kit)

• Design gene-specific primers for SPBC1347.09

• Normalize to reference genes (typically act1+)

Similar studies in fission yeast have identified genes with expression changes during oxidative stress, nutrient starvation, and other conditions, categorizing them into distinct response classes .

What is known about the transcriptional regulation of SPBC1347.09?

Gene regulation in S. pombe involves several documented pathways:

Regulatory mechanisms to investigate:

• Stress response pathways: The Pap1-Oxs1 pathway regulates numerous genes during disulfide stress

• Nutrient response: Genes show distinct expression patterns during phosphate starvation

• RNA quality control: Nonsense-mediated decay affects mRNA stability of many transcripts

To study SPBC1347.09 regulation:

• Analyze its promoter region for binding motifs of known transcription factors

• Perform ChIP experiments to identify proteins binding to its promoter

• Test expression in strains lacking specific transcription factors

• Examine transcription during cell cycle progression and stress conditions

How can I study SPBC1347.09 in the context of protein interaction networks?

Protein interaction networks provide valuable insights into protein function:

Network analysis approaches:

• Protein-Protein Interaction (PPI) Networks:

  • Map direct interactions using yeast two-hybrid or Co-IP/MS

  • Identify protein complexes containing SPBC1347.09

• Co-Expression Networks:

  • Analyze transcriptome data to identify genes with expression patterns similar to SPBC1347.09

  • Construct correlation networks from RNA-Seq data across various conditions

• Genetic Interaction Networks:

  • Generate double mutants combining SPBC1347.09Δ with other deletions

  • Analyze synthetic lethality or fitness defects

  • Use high-throughput approaches like synthetic genetic arrays (SGAs)

What approaches can be used to determine SPBC1347.09 subcellular localization?

Determining subcellular localization provides crucial functional insights:

Recommended methods:

Protocol elements:

• Generate strain with SPBC1347.09-GFP or prepare antibody for immunofluorescence

• For GFP visualization, image live cells directly

• For immunofluorescence:

  • Fix cells with formaldehyde

  • Permeabilize cell wall using enzymatic digestion

  • Incubate with primary antibody followed by fluorescent secondary antibody

  • Image using confocal or fluorescence microscopy

How can SPBC1347.09 antibody be used for comparative studies across different yeast species?

Cross-species studies can provide evolutionary insights:

Approach:

• Assess sequence conservation between SPBC1347.09 and homologs in other species

• Test antibody cross-reactivity by Western blot against extracts from multiple yeast species

• For species with confirmed cross-reactivity, compare:

  • Expression levels under matched conditions

  • Subcellular localization

  • Co-purifying partners

Important considerations:

• Epitope conservation is critical for cross-reactivity

• Even with high sequence similarity, antibody binding may vary

• Include positive controls (conserved proteins like tubulin) for each species

• Consider raising antibodies against conserved peptide sequences for better cross-species recognition

How can I troubleshoot non-specific binding when using SPBC1347.09 antibody?

Non-specific binding is a common challenge:

Troubleshooting matrix:

Optimization steps:

• Titrate antibody concentration (1:1,000 to 1:20,000)

• Test different blocking agents (5% milk, 5% BSA)

• Increase washing stringency (add 0.1-0.3% Tween-20)

• Pre-adsorb antibody with extract from SPBC1347.09Δ strain

What controls should be included in SPBC1347.09 expression or localization studies?

Rigorous controls ensure reliable results:

Essential controls:

• Negative genetic control: SPBC1347.09 deletion strain

• Positive expression control: Known constitutive protein (e.g., tubulin)

• Subcellular markers: Co-staining with known compartment markers

• Experimental controls:

  • For stress response: Non-stressed condition

  • For developmental studies: Different cell cycle stages

  • For protein interactions: Reciprocal tagging of interaction partners

Recommended validation approach:

• Combine multiple detection methods (e.g., fluorescence microscopy and biochemical fractionation)

• Use different antibodies or epitope tags where possible

• Include biological replicates to assess variability