SPBC1347.14c Antibody

What is SPBC1347.14c and what is its role in S. pombe?

SPBC1347.14c is a gene in fission yeast that belongs to a specific class of stress-responsive genes. Based on RNA-seq and qRT-PCR analyses, it is categorized as a Class II stress response gene along with sro1+ and SPAC23D3.12. These genes show a distinctive expression pattern during disulfide stress conditions, particularly when cells are exposed to diamide. The expression of SPBC1347.14c is unaffected by single deletion of either pap1Δ or oxs1Δ, but shows elevated expression in the pap1Δoxs1Δ double mutant background . This suggests that either Oxs1 or Pap1 alone is sufficient to repress SPBC1347.14c transcription, and that de-repression requires loss of both proteins. The unique regulatory pattern makes SPBC1347.14c an important gene for understanding transcriptional repression mechanisms during stress response in S. pombe.

Why are antibodies against SPBC1347.14c important for research?

Antibodies against SPBC1347.14c are valuable research tools for several reasons:

• They enable direct investigation of SPBC1347.14c protein expression, localization, and post-translational modifications during various stress conditions

• They facilitate chromatin immunoprecipitation (ChIP) experiments to study the regulation of this gene by transcription factors like Pap1 and Oxs1

• They allow researchers to track SPBC1347.14c protein in co-immunoprecipitation experiments to identify interacting partners

• They provide a means to validate genetic findings with protein-level evidence, particularly important when studying stress response pathways where post-translational regulation may be significant

Since SPBC1347.14c shows a unique expression pattern during disulfide stress, antibodies are crucial for understanding its regulation at the protein level beyond transcriptional changes observed in RNA analyses.

What types of SPBC1347.14c antibodies are most useful for S. pombe research?

For S. pombe research involving SPBC1347.14c, several antibody types are particularly useful:

• Anti-epitope tag antibodies: When working with tagged versions of SPBC1347.14c (HA, FLAG, GFP, etc.), commercially available high-specificity antibodies against these tags are preferred. The search results mention successful use of anti-HA antibodies (Cat# ab9110, Abcam) and anti-FLAG antibodies (Cat# F1804, Sigma) in immunoprecipitation studies in S. pombe .

• Custom antibodies against SPBC1347.14c: These are raised against purified SPBC1347.14c protein or synthetic peptides derived from its sequence. These are essential when studying the native, untagged protein.

• ChIP-grade antibodies: These must meet higher stringency requirements for chromatin immunoprecipitation experiments when studying the regulation of SPBC1347.14c or its role as a potential transcriptional regulator.

When selecting antibodies for SPBC1347.14c research, considerations should include specificity, sensitivity in detecting both native and denatured forms of the protein, and compatibility with desired applications (Western blotting, immunoprecipitation, ChIP, immunofluorescence).

What is the recommended protocol for SPBC1347.14c antibody validation?

Proper antibody validation is critical for ensuring experimental reliability. For SPBC1347.14c antibodies, the recommended validation protocol includes:

• Specificity testing: Compare wild-type strains with SPBC1347.14c deletion strains to confirm absence of signal in the latter. This approach mirrors the validation method used for anti-Oxs1 antibodies described in the search results, where signal specificity was confirmed by showing reduced binding in an oxs1Δ strain .

• Western blot validation: Confirm the antibody detects a band of the expected molecular weight. For tagged versions of SPBC1347.14c, parallel detection with both anti-tag and anti-SPBC1347.14c antibodies should yield identical banding patterns.

• Immunoprecipitation validation: Following the protocols described in the search results for other S. pombe proteins, immunoprecipitate SPBC1347.14c and verify by mass spectrometry or western blotting .

• ChIP validation: If the antibody will be used for ChIP studies, verify enrichment at known target loci compared to control regions and confirm this enrichment is lost in SPBC1347.14c deletion strains.

• Cross-reactivity assessment: Test against closely related proteins or in heterologous expression systems to confirm specificity.

Each validation step should include appropriate controls as described in the research literature for other S. pombe proteins.

How should ChIP experiments be designed and optimized for SPBC1347.14c studies?

Based on the ChIP protocols described in the search results for similar S. pombe studies, the following considerations are important for SPBC1347.14c ChIP experiments:

• Cell preparation: Grow S. pombe cells to OD600 0.5 and expose to appropriate stress conditions (e.g., 1.5 mM diamide for studying disulfide stress) .

• Crosslinking and lysis: Crosslink with formaldehyde and lyse cells in appropriate buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NonidetP-40, 0.1% SDS, 12 mM sodium deoxycholate) using glass beads in a bead beater (Fastprep-24 MP Biomedical) .

• Chromatin shearing: Release and shear chromatin using a sonicator (M220, Covaris) to obtain fragments of 200-500 bp .

• Immunoprecipitation: Use 5 μg of antibody per sample with overnight incubation at 4°C, followed by capture with protein A/G beads (e.g., Dynabeads mixture) .

• Washing and elution: Wash beads thoroughly (at least 6 times) with buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% NonidetP-40, 5% glycerol pH 7.4) and elute DNA .

• qPCR analysis: Design primers targeting the promoter regions of SPBC1347.14c and control regions. Control primers should target either coding regions of unrelated genes (e.g., gpd3+) or intergenic regions .

• Data normalization: Express results as enrichment relative to input and include a no-antibody control to account for background binding.

For studying the regulation of SPBC1347.14c by Pap1 and Oxs1, parallel ChIP experiments with antibodies against these transcription factors would be informative.

What are the best methods for studying SPBC1347.14c expression during stress responses?

Based on the experimental approaches described in the search results, the following methodologies are recommended for studying SPBC1347.14c expression during stress responses:

• RNA analysis:

  • Grow cells to OD600 ~0.3

  • Treat with appropriate stressor (e.g., 1.5 mM diamide)

  • Collect samples at multiple time points (0-5 hours)

  • Extract RNA using RNeasy or similar kit

  • Perform RT-qPCR normalizing to act1+ as an internal control

• Protein analysis:

  • Western blotting with SPBC1347.14c-specific antibodies

  • Co-immunoprecipitation to identify interaction partners during stress

  • Subcellular fractionation to determine localization changes

• Live cell imaging:

  • Generate GFP-tagged SPBC1347.14c strains

  • Monitor localization changes during stress using fluorescence microscopy

  • Consider dual labeling with markers for specific cellular compartments

• Genetic analysis:

  • Compare wild-type, single mutants (pap1Δ, oxs1Δ), and double mutant (pap1Δoxs1Δ) backgrounds

  • Use complementation studies with plasmid-expressed SPBC1347.14c

For comprehensive analysis, a time-course experiment capturing both early and late responses to stress is recommended, as the search results indicate complex temporal regulation of stress response genes in S. pombe .

How can protein-protein interactions involving SPBC1347.14c be effectively studied?

Based on the interaction studies described in the search results for related proteins, the following methods are recommended for studying SPBC1347.14c interactions:

• Co-immunoprecipitation (Co-IP):

  • Grow cells to OD600 0.5 and apply appropriate stress conditions

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

  • Immunoprecipitate with anti-SPBC1347.14c antibody or anti-tag antibody

  • Detect interacting partners by western blotting or mass spectrometry

• GST pull-down assays:

  • Clone SPBC1347.14c into pGEX-4T-1 to generate GST-fusion protein

  • Express in E. coli BL21(DE3)pLysS

  • Purify using MagneGST™ beads

  • Incubate with cell lysates or purified candidate interacting proteins

  • Wash extensively and analyze by western blotting

• Yeast two-hybrid screening:

  • Use SPBC1347.14c as bait to screen a S. pombe cDNA library

  • Validate positive interactions with Co-IP or pull-down assays

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse SPBC1347.14c and candidate partners to complementary fragments of a fluorescent protein

  • Monitor interaction by reconstitution of fluorescence in vivo

• Proximity-dependent biotin labeling (BioID or TurboID):

  • Fuse SPBC1347.14c to a biotin ligase

  • Identify proteins in proximity through streptavidin pull-down and mass spectrometry

When designing interaction studies, consider both normal and stress conditions, as SPBC1347.14c's interactions may change significantly during the stress response.

How does the regulation of SPBC1347.14c differ from other stress response genes in S. pombe?

The regulation of SPBC1347.14c displays unique characteristics compared to other stress response genes in S. pombe:

SPBC1347.14c belongs to Class II genes, which show a unique pattern where:

• Expression is unaffected by single deletion of either pap1+ or oxs1+

• Expression is elevated in the double mutant pap1Δoxs1Δ

• The regulatory mechanism suggests that either Oxs1 or Pap1 alone is sufficient to repress transcription

• De-repression requires loss of both proteins

This pattern contrasts with Class I genes (which show additive positive regulation by Pap1 and Oxs1) and Class III genes (where Oxs1 prevents Pap1-mediated repression). The distinct regulatory pattern of SPBC1347.14c makes it an interesting model for studying redundant repression mechanisms during stress response.

What approaches should be used to resolve contradictory findings about SPBC1347.14c function?

When faced with contradictory findings regarding SPBC1347.14c function, researchers should consider the following systematic approaches:

• Validate strain backgrounds: Ensure genetic backgrounds are correctly validated, as phenotypic differences can arise from secondary mutations. As noted in the search results for septins, "controversial results" can emerge from different strain backgrounds .

• Control for experimental conditions: Standardize growth conditions, stress parameters, and timing of measurements. The search results emphasize that starvation timing and conditions significantly impact cellular responses .

• Use multiple methodological approaches:

  • Combine genetic approaches (gene deletion, overexpression)

  • Validate with biochemical methods (protein levels, post-translational modifications)

  • Confirm with cell biological approaches (localization, interaction studies)

  • Support with systems-level data (transcriptomics, proteomics)

• Consider context-dependent functions: SPBC1347.14c may have different roles depending on:

  • Cell cycle stage

  • Nutritional status

  • Presence of different stressors

  • Genetic background dependencies

• Quantitative assessments: Use standardized quantitative methods to describe cellular states, as suggested in the search results regarding cytoplasmic freezing: "How to quantitatively describe the cytoplasmic state of cells during starvation" .

• Systematic mutation analysis: Generate point mutations or domain deletions to dissect the specific functional regions of SPBC1347.14c.

When publishing or presenting contradictory findings, clearly describe all experimental conditions and methodological details to enable proper comparison between studies.

What are common pitfalls when working with SPBC1347.14c antibodies and how can they be avoided?

Based on the experimental protocols described in the search results, researchers should be aware of these common pitfalls when working with S. pombe proteins like SPBC1347.14c:

• Non-specific binding:

  • Problem: High background signal in western blots or immunoprecipitation experiments

  • Solution: Optimize antibody concentration, increase washing stringency, and include additional blocking agents (5% milk or BSA)

• Poor immunoprecipitation efficiency:

  • Problem: Low yield of target protein in immunoprecipitation experiments

  • Solution: Follow the optimized protocol described in the search results using Dynabeads® Protein A:Protein G mixture (1:1) and extend incubation times at 4°C

• Variability in stress response:

  • Problem: Inconsistent expression changes of SPBC1347.14c across experiments

  • Solution: Standardize culture conditions strictly and collect time-course samples to capture the dynamic response

• Cross-reactivity with related proteins:

  • Problem: Antibody recognizes proteins other than SPBC1347.14c

  • Solution: Include appropriate controls (SPBC1347.14c deletion strains) and perform validation by mass spectrometry

• Fixation-related epitope masking:

  • Problem: Loss of antibody recognition in fixed cells for immunofluorescence

  • Solution: Test multiple fixation protocols (formaldehyde, methanol, or combined)

• Stress-dependent protein modifications:

  • Problem: Stress conditions alter post-translational modifications affecting antibody recognition

  • Solution: Use multiple antibodies targeting different epitopes or use tagged versions of SPBC1347.14c

• Poor ChIP efficiency:

  • Problem: Low enrichment in ChIP experiments

  • Solution: Optimize crosslinking time, sonication conditions, and antibody amount following the protocol described for similar experiments with fission yeast proteins

Maintain strict temperature control throughout all procedures, as the search results emphasize keeping samples cold (in ice/water) during processing .

How should data from SPBC1347.14c experiments be normalized and analyzed?

Based on the analytical approaches described in the search results for similar gene expression studies in S. pombe, the following normalization and analysis strategies are recommended:

When comparing SPBC1347.14c regulation across different conditions or genetic backgrounds, maintain consistent normalization strategies throughout the analysis to ensure valid comparisons.

What quality control measures are essential when preparing SPBC1347.14c fusion proteins for antibody production?

Based on the protein purification and fusion protein protocols described in the search results, the following quality control measures are essential when preparing SPBC1347.14c fusion proteins for antibody production:

• Sequence verification:

  • Confirm the complete coding sequence of SPBC1347.14c is correctly cloned in-frame with the fusion tag (GST, His, etc.)

  • Verify absence of mutations or frameshifts that could alter the protein's epitopes

• Expression optimization:

  • Test multiple expression conditions (temperature, IPTG concentration, induction time)

  • Compare expression in different E. coli strains (the search results mention successful use of BL21(DE3)pLysS)

  • Confirm expression by SDS-PAGE and western blotting

• Solubility assessment:

  • Analyze distribution between soluble and insoluble fractions

  • Optimize lysis conditions to maximize soluble protein yield

  • Consider adding solubility enhancers (lower temperature, co-expression with chaperones)

• Purification quality:

  • Monitor purification by SDS-PAGE at each step

  • Assess purity through Coomassie staining (aim for >90% purity)

  • Confirm identity by western blotting and/or mass spectrometry

  • Test binding activity using functional assays where applicable

• Protein integrity:

  • Analyze for degradation products using western blotting

  • Perform dynamic light scattering to assess aggregation state

  • Verify correct folding using circular dichroism or fluorescence spectroscopy

• Endotoxin removal:

  • Include endotoxin removal steps for proteins intended for antibody production

  • Quantify residual endotoxin levels using LAL assay

• Epitope accessibility:

  • If producing antibodies against specific domains, confirm these regions are exposed in the fusion protein

  • Consider using both N-terminal and C-terminal fusion constructs to maximize epitope diversity

Following the GST fusion protein methodology described in the search results, including proper buffer composition (25 mM Tris–HCl pH 7.2, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NonidetP-40, 1 mM DTT), will help ensure high-quality fusion protein preparation .

What emerging technologies could enhance SPBC1347.14c antibody-based research?

Several emerging technologies could significantly advance research involving SPBC1347.14c antibodies:

• Proximity labeling technologies:

  • BioID, TurboID, or APEX2 fusions to SPBC1347.14c could identify proximal interaction partners in living cells

  • These approaches would complement traditional co-immunoprecipitation methods by capturing transient or weak interactions

• Single-cell protein analysis:

  • Antibody-based techniques like Fluidigm CyTOF or REAP-seq could reveal cell-to-cell variability in SPBC1347.14c expression

  • This would be particularly valuable for understanding heterogeneous responses to stress conditions

• Super-resolution microscopy:

  • Techniques like STORM, PALM, or STED using fluorescently-labeled antibodies could provide nanoscale resolution of SPBC1347.14c localization

  • This could reveal previously undetectable subcellular organization patterns

• Nanobodies and single-domain antibodies:

  • Smaller antibody fragments could provide better access to epitopes in crowded cellular environments

  • These may be particularly useful in cytoplasmic freezing (CF) conditions described in the search results

• Intrabodies for live-cell tracking:

  • Antibody fragments expressed intracellularly could track SPBC1347.14c dynamics in living cells

  • This approach would overcome limitations of fixing cells or using fusion proteins

• CUT&Tag or CUT&RUN techniques:

  • These could provide higher resolution mapping of SPBC1347.14c interactions with chromatin compared to traditional ChIP

  • They require less starting material and offer better signal-to-noise ratios

• Spatial transcriptomics combined with protein detection:

  • Technologies like MERFISH combined with immunofluorescence could correlate SPBC1347.14c protein localization with gene expression patterns at the single-cell level

These technologies would be particularly valuable for understanding SPBC1347.14c's role during the cytoplasmic transitions described in the search results, such as the liquid-to-solid transition during stress responses .

How might understanding SPBC1347.14c function contribute to broader knowledge about stress responses?

Understanding SPBC1347.14c function could contribute to broader knowledge about stress responses in several significant ways:

• Insight into redundant transcriptional repression mechanisms:

  • SPBC1347.14c's unique regulatory pattern (repressed by either Pap1 or Oxs1 alone) represents an interesting model of redundant repression

  • This could reveal principles of transcriptional network robustness applicable across species

• Connections between disulfide stress and other cellular processes:

  • SPBC1347.14c is regulated specifically during disulfide stress (diamide exposure, Cd treatment)

  • Understanding its function could reveal previously unknown connections between redox homeostasis and other cellular pathways

• Evolutionary conservation of stress response mechanisms:

  • The search results indicate conservation of Oxs1-Pap1 interactions across species (human, Arabidopsis, S. pombe)

  • Determining if SPBC1347.14c function is similarly conserved could reveal fundamental stress response mechanisms

• Cytoplasmic state transitions during stress:

  • SPBC1347.14c could play a role in the cytoplasmic freezing (CF) phenomenon described in the search results

  • This might contribute to understanding how cells maintain anisotropic organization during energy-limited conditions

• Integration of multiple stress response pathways:

  • As a gene regulated by two transcription factors (Pap1 and Oxs1), SPBC1347.14c sits at the intersection of multiple signaling pathways

  • This position makes it valuable for understanding pathway crosstalk and integration

• Model for conditional gene regulation:

  • The complex regulation of SPBC1347.14c (repressed by either Pap1 or Oxs1) represents an interesting regulatory logic

  • This could serve as a model for understanding similar regulatory circuits in other organisms

• Potential applications to cellular adaptation in biotechnology:

  • Understanding SPBC1347.14c's role in stress adaptation could inform strategies for engineering stress-resistant cells for biotechnological applications

The search results indicate that stress response genes like SPBC1347.14c contribute to fitness in challenging environments , highlighting the broader significance of understanding these regulatory mechanisms.

What are the most promising genetic approaches to further investigate SPBC1347.14c function?

Based on the genetic approaches described in the search results and current trends in molecular genetics, the following genetic approaches would be most promising for further investigating SPBC1347.14c function:

• CRISPR-based approaches:

  • CRISPR interference (CRISPRi) for tunable repression of SPBC1347.14c

  • CRISPR activation (CRISPRa) for enhanced expression

  • Base editing for introducing specific point mutations without double-strand breaks

  • Prime editing for precise insertions or deletions

• Synthetic genetic array (SGA) analysis:

  • Systematic creation of double mutants combining SPBC1347.14c deletion with other non-essential genes

  • This would reveal genetic interactions and functional relationships

  • The search results mention successful application of genetic interaction screens in S. pombe

• Anchor-away system:

  • Rapid conditional depletion of SPBC1347.14c protein from specific cellular compartments

  • This would help determine in which cellular location SPBC1347.14c function is required

• Degron tagging:

  • Addition of auxin-inducible or temperature-sensitive degron tags to SPBC1347.14c

  • This would allow rapid protein depletion to study acute effects versus adaptation responses

• Domain-specific mutations:

  • Systematic mutagenesis of predicted functional domains

  • Complementation analysis with mutated versions to identify essential regions

• Promoter replacement:

  • Replacing the native SPBC1347.14c promoter with regulatable promoters (e.g., nmt1)

  • This would allow studying the effects of constitutive expression or overexpression

• Heterologous expression:

  • Expression of SPBC1347.14c orthologs from other species in S. pombe

  • The search results describe successful cross-species complementation studies for related genes

• Quantitative fitness analysis (QFA):

  • The search results describe QFA as a powerful approach to determine how genetic backgrounds impact fitness in different environments

  • This could be applied to SPBC1347.14c to understand its contribution to fitness under various stress conditions

These approaches, particularly when combined, would provide complementary insights into SPBC1347.14c function, regulation, and its role in stress response pathways.