SPAC4G8.04 Antibody

What is SPAC4G8.04 and why is it studied in research?

SPAC4G8.04 is a TBC domain-containing protein found in Schizosaccharomyces pombe (fission yeast). TBC domain proteins typically function as GTPase-activating proteins (GAPs) for Rab GTPases, playing crucial roles in membrane trafficking pathways. Studying this protein helps elucidate fundamental cellular processes in eukaryotic cells. Researchers investigate SPAC4G8.04 to understand conserved mechanisms of vesicular transport, which have implications for understanding similar processes in higher eukaryotes including humans.

What applications is the SPAC4G8.04 antibody validated for?

The SPAC4G8.04 antibody has been validated primarily for detecting the endogenous protein in fission yeast samples. Common applications include:

• Western blotting

• Immunocytochemistry (ICC)

• Immunofluorescence (IF)

• Immunoprecipitation (IP)

When using this antibody in novel applications, thorough validation steps should be conducted including appropriate controls to ensure specificity and sensitivity in your experimental system .

What are the key differences between monoclonal and polyclonal antibodies against SPAC4G8.04?

For highly conserved domains like the TBC domain in SPAC4G8.04, monoclonal antibodies generally offer better specificity but may be more sensitive to epitope masking or denaturation depending on experimental conditions .

What are the optimal conditions for Western blot detection of SPAC4G8.04?

For optimal Western blot detection of SPAC4G8.04:

• Sample preparation:

  • Extract proteins under non-denaturing conditions if targeting conformational epitopes

  • Include protease inhibitors to prevent degradation

• Gel electrophoresis:

  • Use 10-12% polyacrylamide gels for optimal separation

  • Expected molecular weight: Approximately 30-35 kDa

• Transfer and blocking:

  • PVDF membranes typically yield better results than nitrocellulose

  • Block with 5% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20)

• Antibody incubation:

  • Primary antibody dilution: 1:1000 to 1:2000

  • Incubate overnight at 4°C for optimal binding

  • Secondary antibody: Anti-species IgG HRP-conjugate at 1:5000 dilution

• Detection:

  • Enhanced chemiluminescence (ECL) substrate

  • Exposure time: Start with 30 seconds, adjust as needed

Validation through knockout or knockdown controls is essential to confirm specificity .

How should researchers optimize immunofluorescence protocols for SPAC4G8.04 detection in fission yeast?

Optimizing immunofluorescence for SPAC4G8.04 in S. pombe requires specific considerations:

• Cell fixation:

  • 4% paraformaldehyde (15 minutes at room temperature) preserves cellular architecture

  • Alternatively, cold methanol fixation (6 minutes at -20°C) may better preserve certain epitopes

• Cell wall digestion:

  • Critical for antibody penetration

  • Use zymolyase (1 mg/ml for 30 minutes at 37°C)

  • Monitor digestion microscopically to prevent over-digestion

• Permeabilization:

  • 0.1% Triton X-100 (5 minutes) after fixation

  • Over-permeabilization can lead to signal loss and morphological artifacts

• Antibody dilutions:

  • Primary: 1:100 to 1:500 in blocking buffer

  • Secondary: 1:500 fluorophore-conjugated antibody

• Mounting:

  • Use antifade mounting medium containing DAPI for nuclear counterstaining

  • Allow 24 hours for hardening before imaging

• Controls:

  • Include wild-type and SPAC4G8.04 deletion strains in parallel

  • Pre-immune serum control to assess background

Thorough washing between each step is crucial for reducing background fluorescence .

What cross-reactivity concerns exist when using SPAC4G8.04 antibodies in other yeast species?

Cross-reactivity is a significant concern when using S. pombe SPAC4G8.04 antibodies in other yeast species:

• Sequence homology:

  • SPAC4G8.04 shares TBC domain conservation with proteins in:

    • Saccharomyces cerevisiae (budding yeast): ~35-40% homology

    • Candida albicans: ~30% homology

    • Cryptococcus neoformans: ~25% homology

• Epitope conservation:

  • Higher conservation in catalytic regions

  • Lower conservation in regulatory domains

  • Epitope mapping is recommended before cross-species applications

• Validation strategies for cross-species applications:

  • Western blot with recombinant proteins from target species

  • Preabsorption controls with recombinant SPAC4G8.04

  • Peptide competition assays

  • Parallel evaluation with species-specific antibodies when available

• Alternative approaches:

  • Consider epitope-tagging approaches in heterologous systems

  • Use of conserved domain antibodies that target highly preserved regions

Researchers should conduct preliminary validation experiments to determine antibody utility in non-S. pombe species .

How can researchers use SPAC4G8.04 antibodies to study protein-protein interactions in membrane trafficking pathways?

For investigating SPAC4G8.04 protein-protein interactions in membrane trafficking:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in non-denaturing buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, protease inhibitors)

  • Pre-clear lysate with Protein A/G beads

  • Immunoprecipitate with anti-SPAC4G8.04 antibody (5 μg per 1 mg protein)

  • Analyze precipitates for interacting partners by Western blot or mass spectrometry

• Proximity labeling techniques:

  • BioID or TurboID fusion with SPAC4G8.04

  • Expression in S. pombe followed by streptavidin pulldown

  • Mass spectrometry identification of proximal proteins

  • Validation of interactions using SPAC4G8.04 antibody

• Fluorescence microscopy:

  • Dual immunofluorescence with SPAC4G8.04 antibody and antibodies against putative interacting proteins

  • Quantify colocalization using Pearson's or Mander's coefficient

  • Super-resolution microscopy for precise localization

• Förster Resonance Energy Transfer (FRET):

  • Fluorophore-conjugated antibodies against SPAC4G8.04 and potential partners

  • Live cell imaging to detect energy transfer indicating protein proximity

• Split-GFP complementation:

  • Tag SPAC4G8.04 with GFP fragment

  • Tag potential interactors with complementary GFP fragment

  • Fluorescence indicates protein-protein interaction

  • Validate with SPAC4G8.04 antibody in parallel experiments

These approaches reveal functional protein networks involving SPAC4G8.04 in vesicular trafficking pathways .

What strategies can be employed to study post-translational modifications of SPAC4G8.04?

Investigating post-translational modifications (PTMs) of SPAC4G8.04 requires specialized approaches:

• Phosphorylation analysis:

  • Immunoprecipitate SPAC4G8.04 using specific antibody

  • Western blot with phospho-specific antibodies (Ser/Thr/Tyr)

  • Phos-tag SDS-PAGE for mobility shift detection

  • Mass spectrometry for precise phosphorylation site mapping

  • Compare samples with and without phosphatase treatment

• Ubiquitination assessment:

  • Immunoprecipitate under denaturing conditions to preserve ubiquitin linkages

  • Western blot with anti-ubiquitin antibodies

  • Use deubiquitinase inhibitors during sample preparation

  • Consider using tagged ubiquitin (His6-Ub) for pulldown experiments

• SUMOylation detection:

  • Similar to ubiquitination but using SUMO-specific antibodies

  • Include SUMO protease inhibitors (e.g., N-ethylmaleimide)

  • Analyze with anti-SUMO antibodies following SPAC4G8.04 immunoprecipitation

• Glycosylation analysis:

  • Use lectins (e.g., ConA, WGA) for glycoprotein enrichment

  • Treat samples with glycosidases to confirm modifications

  • PNGase F treatment for N-linked glycans

  • O-glycosidase for O-linked glycans

• Acetylation detection:

  • Immunoprecipitate SPAC4G8.04

  • Probe with anti-acetyl-lysine antibodies

  • Include histone deacetylase inhibitors during preparation

• Data integration:

  • Correlate PTM status with cellular conditions (stress, cell cycle phase)

  • Map modifications to protein domains to infer functional significance

  • Compare PTM patterns across growth conditions

These methodologies provide insights into the regulatory mechanisms controlling SPAC4G8.04 function .

How can researchers utilize SPAC4G8.04 antibodies in high-throughput screening approaches?

For all high-throughput approaches, rigorous antibody validation and optimization of detection parameters are essential for reliable results. Appropriate statistical methods should be applied to account for plate-to-plate variation and identify true biological effects versus technical artifacts .

What are common issues when working with SPAC4G8.04 antibodies and how can they be resolved?

For persistent issues, epitope retrieval methods (such as heat-induced or enzymatic retrieval) may improve antibody access to target epitopes. Additionally, switching from polyclonal to monoclonal antibodies (or vice versa) can resolve specificity issues .

How should researchers validate SPAC4G8.04 antibodies for experimental applications?

Comprehensive validation of SPAC4G8.04 antibodies should include:

• Genetic validation:

  • Test antibody against wild-type and SPAC4G8.04Δ (knockout) strains

  • Expected: Signal present in wild-type, absent in knockout

  • If knockout is lethal, use conditional depletion systems

• Expression validation:

  • Test against overexpression systems

  • Correlation between expression level and signal intensity

  • Test in heterologous systems (e.g., E. coli expressing recombinant protein)

• Epitope mapping:

  • Use peptide arrays to identify binding epitopes

  • Confirm epitope conservation if using across species

  • Predict potential cross-reactivity based on epitope sequence

• Specificity assays:

  • Peptide competition experiments

  • Pre-absorption tests with recombinant protein

  • Mass spectrometry confirmation of immunoprecipitated proteins

• Application-specific validation:

  • For Western blotting: Single band at expected molecular weight

  • For immunofluorescence: Subcellular localization consistent with known biology

  • For flow cytometry: Clear separation between positive and negative populations

• Cross-reactivity assessment:

  • Test against related proteins (other TBC domain proteins)

  • Test in multiple cell types/species if intended for broad use

• Documentation:

  • Record all validation experiments with detailed protocols

  • Include representative images/blots in publications

  • Report antibody catalog number, lot, and dilution used

What quality control measurements should be implemented when using SPAC4G8.04 antibodies in quantitative experiments?

For quantitative experiments using SPAC4G8.04 antibodies, implement these quality control measures:

• Standard curve development:

  • Use purified recombinant SPAC4G8.04 protein at known concentrations

  • Generate standard curves for each experiment

  • Determine linear detection range and limits of detection/quantification

• Controls for each experiment:

  • Positive control: Wild-type S. pombe extract

  • Negative control: SPAC4G8.04Δ extract

  • Technical replicates: Minimum of 3 per sample

  • Biological replicates: Minimum of 3 independent cultures/preparations

• Normalization strategy:

  • Include invariant loading controls (e.g., actin, GAPDH)

  • For immunofluorescence: Normalize to cell size or nuclear signal

  • For flow cytometry: Use fluorescence calibration beads

• Signal saturation prevention:

  • Perform dilution series for each new lot of antibody

  • Ensure signal falls within linear range of detection

  • Avoid overexposure in imaging applications

• Batch effects minimization:

  • Process all comparative samples simultaneously

  • Include inter-experimental calibrators

  • Use randomization strategies for sample processing

• Statistical validation:

  • Calculate coefficient of variation (CV) between replicates (aim for <15%)

  • Determine signal-to-noise ratio (S/N >5 for quantitative work)

  • Apply appropriate statistical tests based on data distribution

• Documentation:

  • Record antibody lot, dilution, incubation conditions

  • Document image acquisition parameters

  • Maintain raw data alongside processed results

• Quality indicators table:

These measures ensure reliable quantitative data for publication-quality research using SPAC4G8.04 antibodies .

How should researchers interpret changes in SPAC4G8.04 expression levels across different experimental conditions?

Interpreting SPAC4G8.04 expression changes requires careful consideration of multiple factors:

• Baseline expression context:

  • Establish normal expression across growth phases in wild-type cells

  • Document subcellular localization patterns under standard conditions

  • Consider cell cycle variation (G1, S, G2, M phases)

• Quantification methodology:

  • Western blot: Densitometry normalized to loading controls

  • qPCR: ΔΔCt method with validated reference genes

  • Immunofluorescence: Integrated signal intensity normalized to cell volume

  • Flow cytometry: Mean fluorescence intensity shifts

• Statistical analysis framework:

  • Determine appropriate statistical tests based on data distribution

  • Apply multiple comparison corrections for large datasets

  • Calculate effect sizes in addition to p-values

  • Consider biological significance thresholds (e.g., >1.5-fold change)

• Biological context interpretation:

  • Correlate with membrane trafficking alterations

  • Consider interactions with Rab GTPases

  • Relate to cellular stress responses

  • Evaluate in context of cell growth/division phenotypes

• Comparative analysis:

  • Examine co-regulation with other membrane trafficking genes

  • Compare with related TBC domain proteins

  • Consider parallels with mammalian orthologues

• Validation approaches:

  • Confirm protein level changes with transcript analysis

  • Verify with alternative methodologies

  • Assess functional consequences of observed changes

• Confounding factors to consider:

  • Cell density effects on expression

  • Media composition influences

  • Temperature sensitivity

  • Sample preparation artifacts

A comprehensive interpretation relates expression changes to functional outcomes in membrane trafficking pathways rather than viewing them in isolation .

What are the best practices for analyzing SPAC4G8.04 localization data in cellular compartments?

For robust analysis of SPAC4G8.04 subcellular localization:

• Image acquisition optimization:

  • Use confocal microscopy for optical sectioning

  • Acquire z-stacks to capture complete 3D distribution

  • Select appropriate objective (63x or 100x) for resolution

  • Minimize photobleaching with optimized laser power/exposure

• Co-localization approach:

  • Use established compartment markers:

    • Golgi: Anp1-RFP

    • Endosomes: Vps27-RFP

    • ER: Ero1-RFP

    • Vacuole: FM4-64 staining

  • Calculate quantitative co-localization metrics:

    • Pearson's correlation coefficient (values from -1 to +1)

    • Mander's overlap coefficient (values from 0 to 1)

    • Intensity correlation analysis (ICA)

• Quantitative analysis methods:

  • Line scan analysis across cellular compartments

  • Intensity distribution profiling

  • Object-based co-localization

  • Distance-based metrics between objects

• Dynamic localization assessment:

  • Time-lapse imaging during cellular processes

  • Photobleaching techniques (FRAP/FLIP) to measure mobility

  • Conditional expression systems to monitor transport kinetics

• Software tools for analysis:

  • ImageJ/Fiji with JACoP plugin for co-localization analysis

  • CellProfiler for automated compartment segmentation

  • Imaris for 3D reconstruction and analysis

  • Custom MATLAB scripts for specialized analysis

• Reporting standards:

  • Include scale bars in all images

  • Present representative images alongside quantification

  • Report number of cells analyzed (minimum 30-50 cells per condition)

  • Include raw values for co-localization metrics with statistical analysis

• Functional correlation:

  • Relate localization changes to functional outcomes

  • Test localization under genetic or pharmacological perturbations

  • Connect localization patterns to protein interaction networks

These practices ensure reliable interpretation of SPAC4G8.04 localization data, avoiding common pitfalls such as overinterpretation of partial co-localization or artifacts from sample preparation .

What are promising research areas involving SPAC4G8.04 antibodies in membrane trafficking studies?

Emerging research directions for SPAC4G8.04 in membrane trafficking include:

• Advanced imaging applications:

  • Super-resolution microscopy (STORM, PALM) for precise localization

  • Lattice light-sheet microscopy for dynamic trafficking events

  • Correlative light-electron microscopy (CLEM) to connect function with ultrastructure

  • Live-cell tracking of SPAC4G8.04 with split-fluorescent protein technologies

• Disease model applications:

  • Investigation of SPAC4G8.04 orthologs in neurodegenerative disorders

  • Cancer cell trafficking alterations linked to TBC domain proteins

  • Trafficking dynamics in models of secretory/lysosomal storage diseases

  • Therapeutic targeting of dysfunctional membrane trafficking pathways

• Synthetic biology approaches:

  • Engineered SPAC4G8.04 variants with modified regulatory domains

  • Optogenetic control of SPAC4G8.04 function

  • Biosensor development for real-time trafficking visualization

  • Synthetic organelle creation with engineered trafficking components

• Systems biology integration:

  • Comprehensive mapping of SPAC4G8.04 interaction network

  • Mathematical modeling of trafficking dynamics

  • Multi-scale modeling from molecular to cellular levels

  • Machine learning applications for trafficking phenotype prediction

• Evolutionary studies:

  • Comparative analysis of TBC domain proteins across eukaryotic lineages

  • Functional conservation testing in diverse species

  • Evolutionary pressures on membrane trafficking systems

  • Ancient origin and specialization of trafficking machinery

These research directions build upon fundamental knowledge of SPAC4G8.04 function while extending into translational applications and theoretical understanding of cellular organization principles .

What technological advancements may improve SPAC4G8.04 detection and functional analysis?

Emerging technologies with potential to transform SPAC4G8.04 research:

• Next-generation antibody technologies:

  • Single-domain nanobodies with improved penetration

  • Recombinant antibody fragments for super-resolution imaging

  • Site-specific conjugation for precise labeling

  • Intrabodies for live-cell detection

• Advanced proteomics approaches:

  • Proximity-dependent biotinylation (BioID, TurboID)

  • Crosslinking mass spectrometry (XL-MS)

  • Thermal proximity coaggregation (TPCA)

  • Limited proteolysis-coupled mass spectrometry

• Genome engineering advances:

  • Prime editing for precise genetic modifications

  • CRISPR interference/activation for endogenous regulation

  • Base editing for specific amino acid modifications

  • Scarless tagging methods for endogenous visualization

• Single-cell technologies:

  • Single-cell proteomics for heterogeneity assessment

  • Spatial transcriptomics to relate location to function

  • Live-cell lineage tracing with protein dynamics

  • Single-cell western blotting for protein analysis

• Microfluidic and organ-on-chip platforms:

  • High-throughput phenotypic screening

  • Real-time monitoring of trafficking dynamics

  • Reconstituted trafficking systems in vitro

  • Gradient generation for directional trafficking studies

• Computational advancements:

  • Deep learning for image analysis and phenotype classification

  • Molecular dynamics simulations of protein interactions

  • Predictive modeling of trafficking network responses

  • Integration of multi-omics data for systems-level understanding

• Technology integration table:

These technological advances will enable increasingly sophisticated analyses of SPAC4G8.04's role in cellular trafficking networks .

How might SPAC4G8.04 research contribute to understanding conserved trafficking mechanisms across eukaryotes?

SPAC4G8.04 research offers valuable insights into evolutionarily conserved trafficking mechanisms:

• Evolutionary conservation analysis:

  • TBC domain proteins represent ancient GTPase regulatory mechanisms

  • Functional conservation despite sequence divergence

  • Core trafficking machinery predates eukaryotic radiation

  • Model for studying essentiality versus adaptatability in trafficking

• Translational research opportunities:

  • Human ortholog identification and functional comparison

  • Conservation of regulatory mechanisms

  • Identification of trafficking vulnerabilities in disease states

  • Therapeutic targeting strategies based on conserved domains

• Comparative genomics framework:

  • Multi-species alignment of trafficking components

  • Gain/loss patterns across evolutionary history

  • Correlation between trafficking complexity and organismal complexity

  • Identification of lineage-specific adaptations

• Structural biology insights:

  • Conservation of catalytic mechanisms

  • Diversification of regulatory domains

  • Evolution of protein-protein interaction interfaces

  • Structure-function relationships across species

• Developmental biology connections:

  • Role of trafficking in cellular differentiation

  • Conservation of trafficking dynamics during development

  • Species-specific adaptations in specialized cell types

  • Evolutionary innovations in trafficking regulation

• Quantitative evolutionary study approaches:

  • Evolutionary rate analysis of trafficking components

  • Positive selection signatures in trafficking pathways

  • Co-evolution networks identifying functional relationships

  • Ancient duplication events leading to specialization

• Phylogenetic distribution of trafficking features: