SPAC4D7.04c Antibody

What is SPAC4D7.04c and why is it important for fission yeast research?

SPAC4D7.04c is a systematic identifier that designates a specific gene in the fission yeast Schizosaccharomyces pombe genome. The gene encodes a protein involved in stress response pathways, particularly within the stress-activated MAPK signaling cascade that controls cytoskeletal dynamics . This protein is significant because it functions within pathways that regulate cellular responses to environmental stressors, similar to how stress-activated protein kinases operate in various eukaryotic organisms.

Research on SPAC4D7.04c contributes to our understanding of fundamental cellular processes like cytoskeletal organization and stress response mechanisms. In S. pombe, proteins involved in these pathways often participate in the assembly and regulation of the contractile actomyosin ring (CAR) during cytokinesis . The study of such proteins provides insights into conserved cellular mechanisms that may be relevant across eukaryotic species, from yeasts to humans.

What detection methods are most effective for visualizing SPAC4D7.04c protein in fission yeast cells?

When visualizing SPAC4D7.04c protein in S. pombe cells, researchers typically employ multiple complementary approaches:

Immunofluorescence microscopy: This technique uses specific antibodies against SPAC4D7.04c to visualize the protein's subcellular localization. For optimal results, cells should be fixed with 3.7% formaldehyde, permeabilized with 1% Triton X-100, and incubated with primary antibodies at 1:500 dilution followed by fluorophore-conjugated secondary antibodies.

GFP-tagging: Creating a genomic integration of GFP-tagged SPAC4D7.04c allows for live-cell imaging of protein dynamics. This approach is particularly valuable for time-lapse experiments tracking protein relocalization during stress responses or cell cycle progression. Similar approaches have been successfully used to track proteins like For3-3GFP during stress responses in S. pombe .

Western blotting: For quantitative analysis of protein levels, western blotting provides reliable detection. Protein extraction using glass bead lysis in the presence of protease inhibitors, followed by SDS-PAGE separation and transfer to PVDF membranes yields consistent results. For optimal detection, use primary antibody at 1:1000 dilution with overnight incubation at 4°C.

How do I optimize antibody specificity for SPAC4D7.04c detection in different experimental applications?

Optimizing antibody specificity for SPAC4D7.04c requires systematic approach:

Pre-absorption with knockout lysates: When available, pre-absorb the antibody with total cell lysates from SPAC4D7.04c deletion strains to remove cross-reactive antibodies. Incubate your antibody preparation with knockout lysate (5mg/ml protein concentration) for 2 hours at room temperature before use.

Titration experiments: Perform systematic titration experiments using different antibody dilutions (1:100 to 1:5000) against wild-type and knockout samples to determine the optimal concentration that maximizes specific signal while minimizing background.

Blocking optimization: Test different blocking agents including 5% BSA, 5% non-fat dry milk, and commercial blocking buffers to identify the condition that minimizes non-specific binding. For S. pombe lysates, 5% BSA often provides superior results due to the unique composition of yeast cell walls.

Validation across techniques: Confirm antibody specificity using multiple detection methods. If an antibody performs well in western blotting but poorly in immunofluorescence, this may indicate epitope masking in fixed cells.

How can I design experiments to investigate SPAC4D7.04c interactions with the SAPK pathway during stress responses?

Investigating SPAC4D7.04c interactions with the SAPK pathway requires sophisticated experimental design:

Epistasis analysis: Generate double and triple mutant strains combining SPAC4D7.04c deletion with mutations in known SAPK pathway components (Sty1, Wis1, Wak1/Win1, Mcs4) to determine genetic interactions . Analyze growth phenotypes under various stress conditions (0.15-0.2 μM LatA, 0.6 M KCl, 1 mM H₂O₂, 40°C) to establish epistatic relationships.

Co-immunoprecipitation under stress conditions: Perform co-IP experiments using GFP-tagged SPAC4D7.04c as bait in both unstressed cells and cells subjected to different stressors. Compare interaction profiles to identify stress-specific binding partners within the SAPK pathway. Include appropriate controls, such as testing in sty1Δ backgrounds to determine Sty1-dependent interactions .

Phosphorylation site mapping: If SPAC4D7.04c is phosphorylated by Sty1 or other SAPK components, identify the phosphorylation sites using mass spectrometry after enriching for phosphopeptides. Create phospho-mutant versions (S/T→A and S/T→E) to test the functional significance of these modifications.

Live-cell microscopy during stress induction: Monitor the dynamic relocalization of fluorescently-tagged SPAC4D7.04c and SAPK components simultaneously using dual-color live-cell imaging during acute stress treatments. This approach can reveal temporal relationships in protein activation and localization changes.

What strategies can resolve contradictory data regarding SPAC4D7.04c's role in cytoskeletal organization?

When facing contradictory data regarding SPAC4D7.04c's cytoskeletal functions:

Strain background analysis: Systematically test whether phenotypic differences arise from genetic background variations. Create clean gene deletions in multiple laboratory strains and analyze phenotypes under identical conditions. Similar approaches have revealed strain-specific differences in stress responses in S. pombe .

Dosage-dependent effects: Examine whether conflicting results stem from different protein expression levels. Create strains with controlled expression using inducible promoters (nmt1-41, nmt1-81) to analyze phenotypes across a range of protein levels.

Conditional alleles: Generate temperature-sensitive or analog-sensitive alleles of SPAC4D7.04c to enable acute protein inactivation, distinguishing between direct and compensatory effects. This approach can reveal immediate consequences of protein loss versus adaptive responses that occur in constitutive mutants.

Cross-species comparison: If contradictory data exists between S. pombe and S. japonicus regarding protein function, conduct parallel experiments in both species under identical conditions. This comparative approach has revealed how the same signaling pathway can have opposite effects on cytoskeletal organization in related species .

How should I analyze SPAC4D7.04c protein levels throughout the cell cycle and in response to environmental stressors?

For comprehensive analysis of SPAC4D7.04c protein dynamics:

Synchronized culture analysis: Establish a synchronization protocol using either centrifugal elutriation or transient cdc25-22 block-and-release. Collect samples at 10-minute intervals across one complete cell cycle. Quantify both total protein levels via western blotting and subcellular localization via microscopy.

Stress response time courses: When examining responses to environmental stressors, implement short sampling intervals (0, 5, 15, 30, 60, 120 minutes) after stress induction. Include controls for both total protein levels and loading (e.g., α-tubulin). This approach has revealed how stress conditions like heat shock (40°C), osmotic stress (0.6 M KCl), and oxidative stress (1 mM H₂O₂) affect protein levels in a SAPK-dependent manner .

Protein half-life determination: Measure protein stability using cycloheximide chase experiments under both normal and stress conditions. This can reveal whether stress-induced changes in protein levels result from altered synthesis or degradation rates, similar to how For3 levels are regulated by SAPK signaling .

Quantitative image analysis: For microscopy data, employ rigorous quantification methods including intensity measurements across defined cellular regions and colocalization analysis with known markers of cellular compartments. Analyze at least 100 cells per condition to account for cell-to-cell variation.

What are the best practices for generating and validating knockout strains for SPAC4D7.04c studies?

Generating reliable SPAC4D7.04c knockout strains requires:

PCR-based gene targeting: Design deletion cassettes with 80-100bp homology arms flanking the entire coding sequence. Use antibiotic resistance markers with different selection markers for verification crosses (kanMX6, natMX6, hphMX6).

Verification protocol: Confirm deletions using:

• Colony PCR with primers external to the targeting cassette

• Genomic DNA PCR with multiple primer combinations

• RT-PCR to verify absence of transcript

• Western blotting to confirm protein absence

• Phenotypic rescue with plasmid-borne wild-type gene

Tetrad analysis: Perform tetrad dissection after crossing the deletion strain to wild-type to verify correct segregation of the marker and associated phenotypes. This ensures the phenotype is linked to the deletion and not caused by secondary mutations.

Reference strain archiving: Maintain multiple verified isolates at -80°C and test for phenotypic consistency between isolates to ensure reproducibility.

How do I resolve non-specific binding issues when using SPAC4D7.04c antibodies in complex lysates?

When facing non-specific binding with SPAC4D7.04c antibodies:

Epitope mapping and antibody selection: If multiple antibodies are available, map the precise epitopes they recognize and select those targeting unique regions. Antibodies recognizing conserved domains often show cross-reactivity with related proteins.

Modified extraction protocols: Test different lysis buffers varying in salt concentration (150-500mM NaCl), detergent type (NP-40, Triton X-100, CHAPS), and additives (glycerol, reducing agents). The addition of 0.1% SDS to blocking solutions can reduce hydrophobic interactions causing non-specific binding.

Two-dimensional analysis: For complex samples, combine conventional SDS-PAGE with isoelectric focusing to improve separation of cross-reactive species. This approach can separate proteins with similar molecular weights but different isoelectric points.

Immunodepletion strategy: Sequentially immunoprecipitate using the antibody until specific targets are depleted, then analyze the remaining pattern of cross-reactivity to identify problematic proteins.

What controls are essential when interpreting protein-protein interaction data involving SPAC4D7.04c?

Essential controls for protein interaction studies include:

Reciprocal co-immunoprecipitation: Perform pull-downs using antibodies against both SPAC4D7.04c and its putative interacting partners. True interactions should be detectable bidirectionally.

Deletion strain controls: Include knockout strains for both SPAC4D7.04c and interaction partners to verify specificity of detected bands.

RNase/DNase treatment: Treat lysates with nucleases prior to immunoprecipitation to eliminate interactions mediated by nucleic acids rather than direct protein-protein contact.

Interaction domain mapping: Create truncation mutants to map specific interaction domains, which provides stronger evidence for direct interactions versus coincidental co-purification.

Crosslinking controls: When using chemical crosslinkers, include concentration gradients and competition with excess unrelated proteins to distinguish specific from non-specific crosslinking.

How do SPAC4D7.04c protein functions compare between S. pombe and other model organisms?

Comparative analysis across species reveals:

Evolutionary conservation: SPAC4D7.04c shows sequence similarity to proteins involved in stress response pathways across fungal species. The highest conservation typically appears in functional domains, while regulatory regions may diverge significantly even between closely related species like S. pombe and S. japonicus .

Functional divergence: Despite sequence conservation, the same signaling pathways can have opposite effects in related species. For example, the SAPK pathway positively regulates cytoskeletal assembly in S. japonicus but negatively controls it in S. pombe . This highlights the importance of experimentally verifying function rather than relying solely on sequence homology.

Cross-species complementation: Experimental evidence from cross-species complementation tests provides the strongest evidence for functional conservation. Systematic testing of whether SPAC4D7.04c homologs from other species can rescue S. pombe mutant phenotypes reveals the extent of functional conservation.

Domain architecture comparison: Analysis of protein domain organization across species can reveal evolutionary changes that correlate with functional divergence, such as acquisition or loss of regulatory domains or binding sites for interacting partners.

What methodological approaches best characterize post-translational modifications of SPAC4D7.04c protein?

For comprehensive PTM analysis:

Mass spectrometry workflow:

• Enrich for SPAC4D7.04c using immunoprecipitation or tandem affinity purification

• Perform parallel digestions with multiple proteases (trypsin, chymotrypsin, Glu-C) to ensure complete coverage

• Employ IMAC or TiO₂ enrichment for phosphopeptides

• Conduct both collision-induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation

• Use label-free quantification to compare PTM levels between conditions

Site-specific antibodies: Develop phospho-specific or other PTM-specific antibodies against modified residues identified by mass spectrometry. These enable monitoring of modification dynamics in response to different stimuli.

Mutagenesis studies: Create point mutations at identified modification sites (phospho-null and phospho-mimetic) to determine functional consequences of these modifications on protein activity, localization, and interactions.

In vitro kinase assays: If phosphorylation is detected, identify the responsible kinases using recombinant proteins and in vitro kinase assays with ATP-γ-³²P. This approach can reveal whether SPAC4D7.04c is a direct substrate of Sty1 or other SAPK pathway components .

How can researchers integrate SPAC4D7.04c antibody data with other -omics approaches for comprehensive pathway analysis?

Integrative multi-omics approaches include:

Proteomics-transcriptomics integration: Correlate changes in SPAC4D7.04c protein levels with transcriptomic data to distinguish between transcriptional and post-transcriptional regulatory mechanisms. This approach revealed that certain proteins like For3 are regulated post-transcriptionally during stress responses .

Interactome dynamics: Combine protein interaction data from co-immunoprecipitation studies with temporal protein abundance measurements to create dynamic interaction networks that change during stress responses or cell cycle progression.

Phosphoproteomics correlation: Integrate SPAC4D7.04c phosphorylation data with global phosphoproteomic datasets to identify co-regulated proteins and place SPAC4D7.04c within signaling networks.

Chromatin association studies: If SPAC4D7.04c has nuclear functions, combine ChIP-seq data with transcriptomics to link its genomic binding sites with gene expression changes, similar to how SRF transcriptional activity is regulated by formin-mediated actin dynamics .

How can high-resolution microscopy techniques enhance our understanding of SPAC4D7.04c dynamics during cytokinesis?

Advanced microscopy approaches provide unprecedented insights:

Super-resolution microscopy: Techniques like structured illumination microscopy (SIM) or photoactivated localization microscopy (PALM) achieve resolution below the diffraction limit (~250nm), enabling precise localization of SPAC4D7.04c relative to the contractile actomyosin ring (CAR) components. This approach has revealed detailed organization of proteins within the S. pombe CAR that was previously unresolvable .

Multi-color 4D imaging: Combining multi-wavelength excitation with time-lapse Z-stack acquisition allows tracking of SPAC4D7.04c together with markers for the mitotic spindle, CAR, and cell membrane throughout cytokinesis. This approach revealed the temporal coordination between mitosis and CAR assembly differs between S. pombe and S. japonicus .

FRAP and photoactivation: These techniques measure protein dynamics, revealing the turnover rate of SPAC4D7.04c at specific subcellular locations. Such experiments can determine whether SPAC4D7.04c exhibits different mobility during normal growth versus stress conditions.

Single-molecule tracking: By labeling only a subset of SPAC4D7.04c molecules, researchers can track individual proteins, measuring diffusion rates and residence times at specific cellular structures. This provides mechanistic insights into how SPAC4D7.04c contributes to dynamic cellular processes.

What approaches can distinguish between direct and indirect effects of SPAC4D7.04c on stress response pathways?

Distinguishing direct from indirect effects requires:

Acute protein inactivation: Use fast-acting approaches like auxin-inducible degron (AID) systems or temperature-sensitive alleles to acutely inactivate SPAC4D7.04c and monitor immediate consequences before compensatory mechanisms engage.

Direct target identification: Employ techniques like BioID or APEX proximity labeling to identify proteins in direct physical proximity to SPAC4D7.04c during normal and stress conditions. This approach can reveal differences in the protein's interaction network across conditions.

In vitro reconstitution: Attempt to reconstitute key reactions with purified components to test for direct biochemical interactions, similar to how direct interactions between For3 and components of the SAPK pathway were characterized .

Temporal resolution: Implement high-temporal-resolution sampling immediately following stress induction (seconds to minutes) to capture primary responses before secondary effects become apparent. This approach revealed that SAPK activation occurs rapidly following cytoskeletal damage .

How should researchers interpret changes in SPAC4D7.04c antibody reactivity following various stress treatments?

When analyzing stress-induced changes in antibody reactivity:

Epitope masking effects: Consider whether stress-induced protein modifications or conformational changes might mask antibody epitopes rather than reflecting actual protein level changes. Use multiple antibodies recognizing different epitopes to distinguish between these possibilities.

Solubility shifts: Stress can alter protein solubility, potentially causing redistribution between soluble and insoluble fractions. Analyze both fractions separately to detect such shifts, as observed with certain cytoskeletal proteins during stress .

Comparative quantification methods: Employ complementary quantification approaches including western blotting, immunofluorescence intensity measurements, and mass spectrometry-based quantification to verify consistent trends across methods.

Post-translational modification controls: Include controls using phosphatase treatment or other modification-removing enzymes to determine whether reactivity changes stem from modifications rather than abundance changes. This approach revealed how stress-activated MAPK signaling modifies target proteins to control cytoskeletal dynamics .