SPAC14C4.07 Antibody

What is SPAC14C4.07 and why is it significant in S. pombe research?

SPAC14C4.07 encodes Sup11p, a protein involved in significant cell wall remodeling processes in Schizosaccharomyces pombe. Research indicates that depletion of Sup11p induces substantial changes in cell wall structure and composition, affecting the expression of numerous glucanases and glucan-related enzymes . The protein appears to be essential for cell viability, making it a critical target for fundamental research in yeast cell biology. Understanding Sup11p function provides insights into cell wall integrity pathways, protein glycosylation processes, and potentially conserved mechanisms across fungal species.

What applications are most suitable for SPAC14C4.07 antibodies?

SPAC14C4.07 antibodies can be utilized across multiple experimental applications, similar to other yeast protein antibodies. Based on established protocols for related research antibodies, the following applications are typically effective:

For all applications, it is recommended to optimize conditions using positive and negative controls as demonstrated in antibody validation protocols for similar S. pombe proteins .

How should spheroplasting be performed when using SPAC14C4.07 antibodies for intracellular detection?

Effective spheroplast preparation is critical when detecting intracellular targets in yeast. For S. pombe cells expressing SPAC14C4.07/Sup11p, follow this optimized protocol:

• Harvest mid-log phase cells (OD600 0.5-0.8) by centrifugation (1000×g, 5 minutes)

• Wash cell pellet with spheroplast buffer (1.2 M sorbitol, 50 mM sodium citrate, pH 5.8)

• Resuspend cells in spheroplast buffer containing 10 mg/ml zymolyase-20T and 5 mM β-mercaptoethanol

• Incubate at 30°C with gentle agitation, monitoring spheroplast formation microscopically

• Stop digestion by adding cold spheroplast buffer

• Centrifuge spheroplasts at reduced speed (400×g, 5 minutes)

• Proceed with subsequent analysis or lysis steps

This protocol has been adapted from established spheroplasting methods for S. pombe as referenced in proteinase K protection assays . Complete digestion of the cell wall is essential for antibody accessibility to intracellular antigens, but over-digestion can compromise cellular integrity and protein detection.

What is the subcellular localization of SPAC14C4.07/Sup11p and how does this impact antibody selection?

Topology analysis of Sup11p (encoded by SPAC14C4.07) indicates it is a luminal-oriented protein anchored via a signal anchor sequence . This membrane association presents specific challenges for antibody detection. When selecting antibodies, consider:

• Epitope accessibility in the luminal domain versus the membrane-anchored region

• Fixation and permeabilization requirements that maintain membrane integrity while allowing antibody access

• Detergent selection for protein extraction that effectively solubilizes membrane proteins without disrupting antibody-epitope interactions

For membrane-associated proteins like Sup11p, antibodies raised against luminal domains typically yield more consistent results in immunofluorescence applications, while antibodies targeting multiple domains may be more effective for denatured applications like Western blotting.

How can post-translational modifications of SPAC14C4.07/Sup11p affect antibody recognition?

Research indicates that Sup11p undergoes O-mannosylation, which significantly impacts protein recognition by antibodies . Additionally, unusual N-glycosylation patterns have been observed in certain mutant backgrounds (e.g., oma4 mutants) . These modifications can alter epitope accessibility and antibody binding in several ways:

• Glycosylation sites may mask epitopes recognized by antibodies

• Conformational changes induced by glycosylation can affect antibody binding efficiency

• Glycosylation patterns may vary under different experimental conditions or genetic backgrounds

When using SPAC14C4.07 antibodies, consider these approaches:

• Use deglycosylation treatments (EndoH treatment) before Western blotting to improve detection

• Compare antibody performance using antibodies targeting different regions of the protein

• Include appropriate controls when working with glycosylation pathway mutants

• Document the specific post-translational state of your protein samples when reporting results

What are the expected molecular weight variations when detecting SPAC14C4.07/Sup11p by Western blot?

Due to post-translational modifications, particularly glycosylation, the apparent molecular weight of Sup11p in SDS-PAGE can differ from its predicted weight based on amino acid sequence. Similar observations have been documented for other glycosylated proteins like the Ly-6B.2 antigen, where N-glycanase treatment reduced the apparent molecular weight from 25-30 kDa to approximately 15 kDa .

For Sup11p, researchers should expect:

• The predicted molecular weight based on amino acid sequence

• Higher apparent molecular weights in wild-type cells due to O-mannosylation

• Altered migration patterns in glycosylation mutants

• Potential shifts after endoglycosidase treatments

Including appropriate size controls and glycosylation mutants in experimental designs helps interpret these variations accurately.

How can SPAC14C4.07 antibodies be used to investigate protein interactions in the secretory pathway?

Investigating Sup11p interactions within the secretory pathway requires specialized approaches due to its membrane localization and role in protein glycosylation. Recommended techniques include:

• Co-immunoprecipitation with membrane solubilization:

  • Use mild detergents (0.5-1% NP-40 or Digitonin) to preserve protein complexes

  • Include protease inhibitors and appropriate buffer conditions

  • Perform reciprocal IPs with antibodies against suspected interaction partners

• Proximity labeling approaches:

  • Express Sup11p fused to BioID or APEX2 enzymes

  • Allow in vivo biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

• Split-reporter systems:

  • Fuse Sup11p to one half of a split fluorescent protein

  • Fuse candidate interacting proteins to the complementary half

  • Monitor for reconstituted fluorescence indicating proximity

These methods can be applied to investigate Sup11p's role in glycosylation pathways and cell wall integrity signaling networks, similar to approaches used for studying protein interactions in related biological processes .

What controls are essential when using SPAC14C4.07 antibodies in transcriptional regulation studies?

When using antibodies to study how SPAC14C4.07/Sup11p expression impacts transcriptional responses, particularly in glycosylation pathways, implement these critical controls:

• Genetic controls:

  • Include sup11+ deletion strains complemented with plasmid-expressed Sup11p

  • Use inducible promoter systems to modulate Sup11p levels

  • Include glycosylation pathway mutants (e.g., oma mutants) as comparative controls

• Technical controls:

  • Validate antibody specificity using epitope-tagged versions and untagged controls

  • Include isotype controls for immunoprecipitation experiments

  • Perform depletion/add-back experiments to confirm specificity of observed effects

• Transcriptional validation:

  • Confirm antibody-based observations with orthogonal methods (RT-qPCR, RNA-seq)

  • Use reporter constructs to validate transcriptional effects

  • Perform chromatin immunoprecipitation to distinguish direct from indirect effects

Microarray analysis has previously demonstrated that Sup11p depletion induces significant transcriptional changes related to cell wall remodeling processes , making these controls essential for accurate data interpretation.

How can redox sensitivity be assessed when working with SPAC14C4.07/Sup11p?

For proteins involved in cell wall processes, redox conditions can significantly impact structure and function. To assess redox sensitivity of Sup11p:

• Use ratiometric measurements with redox-sensitive GFP (roGFP2) fusions:

  • Generate Sup11p-roGFP2 fusion constructs

  • Monitor oxidation states under various cellular conditions

  • Compare results with established redox sensors

• Implement non-reducing versus reducing gel electrophoresis:

  • Prepare samples without and with reducing agents (DTT, β-mercaptoethanol)

  • Compare migration patterns to identify potential disulfide bonds

  • Use thiol-modifying reagents to trap specific redox states

• Perform site-directed mutagenesis of cysteine residues:

  • Systematically mutate cysteine residues in Sup11p

  • Assess functional consequences through phenotypic analysis

  • Determine which cysteines are critical for redox-dependent activities

These approaches can be integrated with ratiometric redox measurements similar to those described for roGFP2 systems in related studies .

What are common issues when detecting SPAC14C4.07/Sup11p in mutant backgrounds?

Detection of Sup11p can be particularly challenging in certain genetic backgrounds, especially glycosylation mutants. Common issues and solutions include:

In oma4 mutant backgrounds specifically, unusual N-glycosylation of Sup11p has been observed , which may require adjusting detection protocols to account for these modifications.

How should experiments be designed to distinguish between direct and indirect effects of SPAC14C4.07/Sup11p depletion?

Distinguishing direct from indirect effects of Sup11p depletion requires carefully designed experiments:

• Temporal analysis:

  • Use time-course experiments with inducible depletion systems

  • Monitor earliest detectable changes following Sup11p depletion

  • Compare with known immediate-early response genes

• Genetic separation of functions:

  • Generate domain-specific mutants of Sup11p

  • Identify separable phenotypes associated with distinct protein domains

  • Use complementation assays with mutant variants

• Direct biochemical validation:

  • Perform in vitro reconstitution of suspected direct activities

  • Use purified components to test specific biochemical functions

  • Implement crosslinking approaches to capture transient interactions

These approaches have been utilized in related studies examining cell wall remodeling processes and protein glycosylation pathways in S. pombe .

What strategies improve the detection of low-abundance SPAC14C4.07/Sup11p in different cellular compartments?

For improved detection of low-abundance Sup11p across cellular compartments:

• Subcellular fractionation enhancement:

  • Implement differential centrifugation optimized for membrane proteins

  • Use density gradient centrifugation to separate specific organelles

  • Verify fraction purity with compartment-specific markers

• Signal amplification techniques:

  • Employ tyramide signal amplification for immunofluorescence

  • Use high-sensitivity detection substrates for Western blotting

  • Consider proximity ligation assays for in situ detection

• Sample enrichment approaches:

  • Perform affinity purification of tagged versions

  • Implement cell synchronization to capture cell-cycle specific expression peaks

  • Use proteasome inhibitors if protein has high turnover rate

These approaches are particularly relevant for membrane proteins like Sup11p that may have restricted localization patterns or expression levels that vary across growth conditions .

How should researchers interpret conflicting results between different detection methods for SPAC14C4.07/Sup11p?

When facing discrepancies between different detection methods:

• First, consider methodological differences:

  • Native versus denaturing conditions affecting epitope accessibility

  • Differential sensitivity to post-translational modifications

  • Variation in detection limits between methods

• Implement validation strategies:

  • Use orthogonal detection methods (e.g., mass spectrometry)

  • Employ multiple antibodies targeting different epitopes

  • Verify with genetically tagged versions of the protein

• Account for biological variables:

  • Cell cycle stage variations in protein expression or modification

  • Growth conditions affecting protein localization or abundance

  • Strain background differences influencing protein processing

Systematic documentation of these variables in experimental reports enables more robust interpretation of apparently conflicting results across different studies.

What statistical approaches are recommended when analyzing quantitative data from SPAC14C4.07 antibody experiments?

For quantitative analysis of Sup11p experiments, consider these statistical approaches:

• For Western blot densitometry:

  • Implement linear range validation for quantification

  • Use ANOVA with post-hoc tests for multiple condition comparisons

  • Include technical and biological replication in experimental design

• For microscopy-based quantification:

  • Apply unbiased sampling approaches (systematic random sampling)

  • Use appropriate colocalization coefficients (Pearson's, Manders')

  • Implement machine learning approaches for complex pattern recognition

• For proteomics integration:

  • Employ appropriate normalization for comparing abundance across samples

  • Use pathway enrichment analysis for related proteins

  • Implement data integration across multiple omics platforms

These statistical approaches should be selected based on experimental design and adjusted for multiple comparisons when appropriate.