SPAC630.04c Antibody

What is SPAC630.04c and why is it studied in research?

SPAC630.04c is a gene in Schizosaccharomyces pombe (fission yeast), coding for a protein of interest in fundamental cellular processes. Research into this protein contributes to our understanding of conserved eukaryotic mechanisms, as S. pombe is a well-established model organism with cellular processes similar to those in higher eukaryotes, including humans .

Methodologically, researchers often use antibodies against SPAC630.04c to:

• Track protein localization during cell cycle phases

• Examine protein expression levels under various conditions

• Study protein-protein interactions in cellular pathways

• Investigate post-translational modifications

What applications are SPAC630.04c antibodies suitable for?

Based on typical polyclonal antibody applications, SPAC630.04c antibodies can be utilized in multiple research techniques:

While specific validation data for this antibody isn't detailed in the available information, these applications follow standard practices for polyclonal antibodies against yeast proteins .

How should samples be prepared for SPAC630.04c detection in S. pombe?

Proper sample preparation is critical for successful detection:

• Cell lysis protocols: For S. pombe, mechanical disruption methods (glass bead beating) in the presence of protease inhibitors are most effective. The cell wall must be completely disrupted while maintaining protein integrity.

• Buffer considerations: Use buffers containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% NP-40, supplemented with protease inhibitor cocktail and phosphatase inhibitors if phosphorylation is being studied.

• Protein denaturation: For SDS-PAGE applications, heat samples to 95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol to ensure proper denaturation.

• Fixation for microscopy: 4% paraformaldehyde fixation for 15-30 minutes is recommended for immunofluorescence in fission yeast, followed by cell wall digestion with zymolyase before antibody incubation .

What controls should be included when using SPAC630.04c antibody?

Rigorous experimental design requires appropriate controls:

• Positive control: Use wild-type S. pombe extract where SPAC630.04c is known to be expressed

• Negative controls:

  • Pre-immune serum (provided with custom antibodies)

  • Samples from SPAC630.04c knockout strains (if available)

  • Secondary antibody only controls to assess non-specific binding

• Loading controls: For Western blots, include detection of housekeeping proteins (α-tubulin, GAPDH) to normalize expression levels

• Specificity controls: Perform peptide competition assays using the immunizing peptide to confirm binding specificity

The pre-immune serum that comes with custom polyclonal antibodies serves as an excellent negative control for experimental validation .

How can epitope mapping be performed to characterize SPAC630.04c antibody binding?

Epitope mapping provides critical information about antibody specificity and can explain cross-reactivity issues:

• Peptide array approach:

  • Generate overlapping peptides (15-20 amino acids with 5-amino acid offsets) spanning the SPAC630.04c protein sequence

  • Spot peptides on cellulose membranes

  • Probe with the antibody using standard immunoblotting protocols

  • Analyze binding patterns to identify epitope regions

• Deletion mutant analysis:

  • Create truncated versions of SPAC630.04c protein

  • Express recombinant fragments in bacteria

  • Perform Western blot analysis to determine which regions are recognized

• Phage display method:

  • Display random peptide libraries on phage

  • Select phages that bind to the SPAC630.04c antibody

  • Sequence selected phages to identify mimotopes

  • Map mimotopes to the SPAC630.04c sequence using bioinformatics tools

These approaches help determine if the antibody recognizes linear or conformational epitopes, which impacts application suitability .

What strategies can overcome cross-reactivity issues with SPAC630.04c antibody?

When cross-reactivity is observed in experimental work:

• Antibody purification strategies:

  • Affinity purification against the specific immunizing peptide

  • Negative selection against known cross-reactive proteins

  • Protein A/G purification to isolate IgG fractions for improved specificity

• Experimental modifications:

  • Increase stringency in washing steps (higher salt concentration, addition of 0.1% SDS)

  • Modify blocking solutions (switch between BSA and milk-based blockers)

  • Pre-absorption with lysates from organisms lacking SPAC630.04c

• Validation approaches:

  • Compare results with alternative antibody clones

  • Use genetic knockouts or knockdowns as specificity controls

  • Perform parallel detection with orthogonal methods (e.g., mass spectrometry)

For Western blotting applications specifically, using milk-based blocking reagents may not be optimal for all antibodies; BSA in PBS or TBS-Tween might provide better results for certain applications, similar to recommendations for other antibodies .

How can SPAC630.04c antibody be used to study protein-protein interactions?

Multiple methodological approaches can be employed:

• Co-immunoprecipitation (Co-IP):

  • Lyse S. pombe cells under non-denaturing conditions

  • Incubate lysate with SPAC630.04c antibody coupled to protein A/G beads

  • Wash extensively to remove non-specific binding

  • Elute bound proteins and analyze by mass spectrometry or Western blot

  • Reciprocal IPs should be performed to confirm interactions

• Proximity-dependent labeling approaches:

  • Create fusion proteins with BioID or APEX2 tags

  • Express in S. pombe

  • Validate expression/localization using SPAC630.04c antibody

  • Perform biotinylation reactions followed by streptavidin pulldown

  • Identify interaction partners by mass spectrometry

• Two-hybrid validation:

  • Verify candidate interactions identified in two-hybrid screens

  • Use SPAC630.04c antibody to confirm expression of hybrid proteins

  • Perform Co-IP to validate interactions in native conditions

The GET3 protein (SPAC1142.06) has been shown to interact with a HIT family protein (SPAC630.04C) in S. pombe through two-hybrid studies, demonstrating the value of antibody-based validation for interaction studies .

What approaches should be used when troubleshooting weak or absent SPAC630.04c signal?

Systematic troubleshooting strategies include:

• Protein expression verification:

  • Determine if SPAC630.04c is expressed under your experimental conditions

  • Consider using RT-PCR to verify transcript expression

  • Test different growth conditions that might affect expression levels

• Sample preparation optimization:

  • Test multiple lysis buffers with different detergent compositions

  • Evaluate the impact of phosphatase/protease inhibitors

  • Consider native vs. denaturing conditions based on application

• Technical adjustments:

  • Antibody concentration titration (5-10 fold range)

  • Extended incubation times (overnight at 4°C)

  • Alternative detection systems (HRP vs. fluorescent secondary antibodies)

  • Enhanced chemiluminescence reagents for Western blotting

• Signal amplification methods:

  • Tyramide signal amplification for immunohistochemistry

  • Biotin-streptavidin systems for enhanced sensitivity

  • Use of high-sensitivity detection reagents like SuperSignal West Femto

If weak signals persist, consider whether post-translational modifications might be affecting epitope accessibility or if the protein is rapidly degraded in your experimental conditions .

How can SPAC630.04c antibody be validated for use in chromatin immunoprecipitation (ChIP) experiments?

Thorough validation is essential before using antibodies in ChIP experiments:

• Specificity assessment:

  • Perform Western blots on whole cell extracts

  • Include controls with overexpressed and depleted SPAC630.04c

  • Verify single band of expected molecular weight

• Optimization protocol:

  • Test multiple fixation conditions (0.5-2% formaldehyde, 5-20 minutes)

  • Evaluate different sonication parameters for optimal chromatin fragmentation

  • Compare various antibody concentrations (2-10 μg per IP)

  • Assess multiple washing stringencies to reduce background

• Positive control loci:

  • Identify genomic regions where SPAC630.04c is known or predicted to bind

  • Design primers for qPCR validation of these regions

  • Include negative control regions (heterochromatin or unexpressed genes)

• Functional validation:

  • Perform ChIP under conditions where SPAC630.04c binding is expected to change

  • Correlate binding patterns with gene expression changes

  • Compare results with published datasets or predictions from motif analysis

The specificity of antibody binding in ChIP applications is particularly critical, as artifactual binding can lead to misinterpretation of protein-DNA interaction data .

What factors should be considered when designing immunofluorescence experiments with SPAC630.04c antibody in fission yeast?

Successful immunofluorescence in yeast requires specific considerations:

• Cell wall removal strategies:

  • Enzymatic digestion with zymolyase (optimal concentration: 100μg/ml for 30 minutes)

  • Spheroplasting efficiency must be monitored by microscopy

  • Buffer osmolarity must be maintained to prevent cell lysis

• Fixation optimization:

  • Paraformaldehyde (3-4%) for protein crosslinking

  • Methanol fixation for cytoskeletal proteins

  • Combination approaches for difficult epitopes

• Permeabilization methods:

  • Triton X-100 (0.1-0.5%) for membrane permeabilization

  • Saponin (0.1%) for milder detergent treatment

  • Duration and temperature optimization critical for balanced permeabilization

• Antibody penetration strategies:

  • Extended incubation times (overnight at 4°C)

  • Use of smaller antibody fragments (Fab) for dense structures

  • Sequential multi-antibody labeling for co-localization studies

• Signal-to-noise optimization:

  • Extensive blocking (3-5% BSA, 5-10% normal serum)

  • Multiple washing steps with increasing stringency

  • Minimizing autofluorescence with sodium borohydride treatment

When performing co-localization studies, careful selection of fluorophores with minimal spectral overlap is essential for accurate interpretation .

How can SPAC630.04c antibody be used to investigate post-translational modifications?

Investigating post-translational modifications (PTMs) requires specialized approaches:

• Modification-specific detection strategies:

  • Use general SPAC630.04c antibody for immunoprecipitation

  • Probe with modification-specific antibodies (phospho, ubiquitin, etc.)

  • Compare migration patterns under conditions that alter modifications

• PTM enrichment methods:

  • Phosphopeptide enrichment using TiO2 or IMAC

  • Ubiquitinated protein enrichment using TUBE technology

  • SUMOylated protein enrichment with SUMO-trap approaches

• Mass spectrometry integration:

  • Immunoprecipitate SPAC630.04c with validated antibody

  • Process for mass spectrometry analysis

  • Use targeted MS methods to identify specific modifications

• Validation experiments:

  • Site-directed mutagenesis of putative modification sites

  • In vitro modification assays with purified enzymes

  • Correlation with cellular conditions known to induce modifications

For ubiquitination studies specifically, techniques similar to those using K63-linkage specific antibodies could be adapted, where the antibody recognizes specific poly-ubiquitin chain configurations .

What considerations are important when using SPAC630.04c antibody across different S. pombe strains?

Strain variation can significantly impact antibody performance:

• Strain-specific protein variation:

  • Sequence polymorphisms may affect epitope recognition

  • Expression levels can vary between laboratory strains

  • Protein localization patterns might differ in mutant backgrounds

• Experimental design requirements:

  • Include strain 972 (reference strain) as control

  • Document strain genotypes comprehensively

  • Measure relative expression levels across strains

• Validation across strains:

  • Verify antibody specificity in each strain background

  • Compare signal intensity and pattern across multiple strains

  • Adjust antibody concentration based on expression differences

• Data interpretation considerations:

  • Account for strain-specific growth rates and cell morphology

  • Consider genetic interactions unique to specific strains

  • Normalize quantitative data appropriately between strains

Most antibodies for S. pombe proteins are typically developed against the reference strain 972 / ATCC 24843, including those for SPAC630.04c, so variations in other strains should be carefully validated .

How should quantitative Western blot analysis be performed with SPAC630.04c antibody?

Rigorous quantitative Western blotting requires:

• Sample preparation standardization:

  • Precise protein quantification (BCA or Bradford assay)

  • Equal loading verified by total protein staining (REVERT, Ponceau S)

  • Consistent sample handling to minimize degradation

• Technical considerations:

  • Include calibration curves with purified protein standards

  • Run biological triplicates minimum (preferably 5+ replicates)

  • Include multiple technical replicates

• Imaging and quantification parameters:

  • Use linear range detection methods (digital imaging)

  • Avoid saturated signals that compromise quantification

  • Implement background subtraction consistently

• Data analysis approach:

  • Normalize to loading controls (total protein preferable to single housekeeping proteins)

  • Apply appropriate statistical tests for comparisons

  • Report both normalized values and standard errors

• Validation methods:

  • Confirm trends with independent biological replicates

  • Verify with orthogonal methods (qPCR, mass spectrometry)

  • Test multiple antibody concentrations to ensure signal linearity

For Western blotting applications, dilution ranges of 1:1000-1:8000 are typically recommended for optimal signal-to-noise ratio with monoclonal antibodies, though specific optimization for polyclonal antibodies against SPAC630.04c may be necessary .

How should contradictory results between different applications using SPAC630.04c antibody be resolved?

When facing discrepancies between techniques:

• Systematic analysis framework:

  • Document exact protocols used for each application

  • Identify variables that differ between successful and unsuccessful applications

  • Test whether epitope accessibility varies in different sample preparation methods

• Antibody characteristics assessment:

  • Determine if the antibody recognizes native vs. denatured epitopes

  • Evaluate if fixation methods affect epitope recognition

  • Consider if protein complexes mask the epitope in certain applications

• Validation strategies:

  • Use alternative antibodies targeting different epitopes

  • Perform epitope mapping to understand binding characteristics

  • Verify results with tagged protein versions (GFP-tagging, FLAG-tagging)

• Technical resolution approaches:

  • Adjust sample preparation to preserve relevant protein states

  • Modify blocking and washing conditions to reduce background

  • Optimize primary and secondary antibody concentrations independently for each application

The structure and accessibility of epitopes can vary dramatically between applications like Western blotting (denatured proteins) and immunofluorescence (fixed native proteins), requiring application-specific optimization .

What strategies can distinguish between specific and non-specific signals when using SPAC630.04c antibody?

Differential analysis methods include:

• Control-based approaches:

  • Compare with signals in knockout/knockdown strains

  • Perform peptide competition assays

  • Pre-absorb antibody with purified antigen

• Signal pattern analysis:

  • Evaluate whether signal distribution matches known biology

  • Assess if signal changes with expected biological perturbations

  • Compare with data from orthogonal detection methods

• Technical discrimination methods:

  • Titrate antibody to minimize background while maintaining specific signal

  • Apply more stringent washing conditions incrementally

  • Test alternative blocking reagents (BSA vs. milk vs. normal serum)

• Molecular weight verification:

  • For Western blots, precise molecular weight determination

  • Use gradient gels for better resolution around target size

  • Consider post-translational modifications that affect migration

Pre-immune serum comparison is particularly valuable for polyclonal antibodies like those against SPAC630.04c, as it provides a direct negative control from the same animal source .

How can researchers determine if SPAC630.04c protein degradation is affecting experimental results?

Protein degradation challenges require systematic investigation:

• Sample preparation optimization:

  • Test multiple lysis buffers with increasing protease inhibitor concentrations

  • Compare flash-freezing vs. direct lysis of samples

  • Evaluate temperature effects during processing (4°C vs. room temperature)

• Degradation assessment methods:

  • Run time-course experiments of sample storage

  • Compare fresh samples with stored samples

  • Look for characteristic degradation patterns (ladder of bands below expected size)

• Stabilization strategies:

  • Add deubiquitinating enzyme inhibitors (PR-619, NEM)

  • Use proteasome inhibitors (MG132) in live cells before harvesting

  • Implement rapid processing workflows with minimal sample handling

• Analytical approaches:

  • Use N- and C-terminal targeted antibodies to identify degradation patterns

  • Perform pulse-chase experiments to measure protein half-life

  • Consider immunoprecipitation followed by mass spectrometry to identify degradation products

Understanding protein degradation mechanisms specific to your experimental system can help distinguish genuine biological regulation from technical artifacts .

What are the best practices for reproducing immunofluorescence localization data with SPAC630.04c antibody?

Rigorous reproducibility standards include:

• Protocol standardization:

  • Document complete fixation and permeabilization parameters

  • Specify exact antibody dilutions, incubation times and temperatures

  • Record image acquisition settings (exposure, gain, offset)

• Controls for each experiment:

  • Include no-primary antibody controls

  • Use pre-immune serum controls

  • Process wild-type and negative control samples in parallel

• Quantification approaches:

  • Analyze multiple cells (>100) across different fields

  • Apply consistent thresholding methods for signal detection

  • Use automated analysis pipelines to reduce bias

• Validation requirements:

  • Confirm patterns with alternative fixation methods

  • Verify localization with orthogonal approaches (e.g., GFP tagging)

  • Test antibody specificity by competition with immunizing peptide

• Data presentation standards:

  • Show representative images alongside quantification

  • Include scale bars and indicate image processing techniques

  • Present images from multiple independent experiments

For immunofluorescence applications, dilution ranges of 1:50-1:200 are typically recommended as starting points, though optimization for specific experimental conditions is essential .

How can researchers integrate SPAC630.04c antibody data with other omics datasets?

Multi-omics integration strategies:

• Data correlation approaches:

  • Compare protein levels (Western blot) with transcriptomics data

  • Correlate protein localization changes with phosphoproteomics

  • Link protein-protein interactions with genetic interaction networks

• Temporal integration methods:

  • Align time-course experiments across different data types

  • Account for delays between transcription, translation, and modifications

  • Develop integrated models of regulatory dynamics

• Functional analysis frameworks:

  • Map antibody-derived protein data to pathway databases

  • Overlay protein interaction data with metabolomics changes

  • Use protein localization information to constrain network models

• Visualization and analysis tools:

  • Cytoscape for network visualization and analysis

  • MultiOmics Factor Analysis for dimension reduction across data types

  • Perseus for proteomics and antibody-based data integration

• Validation pipeline:

  • Design targeted experiments to test hypotheses from integrated analysis

  • Use CRISPR-based approaches to verify key regulatory relationships

  • Apply mathematical modeling to predict system behavior