SPBC8E4.01c Antibody

What is SPBC8E4.01c and why is it important in fission yeast research?

SPBC8E4.01c (also known as SPBP4G3.01) encodes a putative inorganic phosphate transporter in Schizosaccharomyces pombe . The protein is predicted to play a crucial role in phosphate homeostasis, which is essential for numerous cellular processes including signal transduction, energy metabolism, and nucleic acid synthesis. Studying this transporter contributes to our understanding of nutrient acquisition in unicellular eukaryotes and phosphate transport mechanisms that may be conserved across species.

What types of SPBC8E4.01c antibodies are currently available for research?

Currently, rabbit polyclonal antibodies against SPBC8E4.01c are commercially available. These antibodies are specifically designed to recognize SPBC8E4.01c in Schizosaccharomyces pombe (strain 972/24843) . The antibodies undergo antigen-affinity purification to ensure specificity and are validated for applications such as Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA) . Researchers should verify the isotype (typically IgG) and host species when selecting an antibody for their specific experimental needs.

What are the recommended applications for SPBC8E4.01c antibodies?

SPBC8E4.01c antibodies have been validated primarily for Western blotting (WB) and ELISA applications . For Western blotting, these antibodies allow researchers to detect and quantify SPBC8E4.01c protein expression levels in cell lysates. ELISA applications permit quantitative measurement of the protein in solution. While not explicitly validated for other techniques, researchers may explore additional applications such as immunoprecipitation, immunofluorescence, or chromatin immunoprecipitation with appropriate controls to ensure antibody specificity in these contexts.

How should I design controls when using SPBC8E4.01c antibody in Western blot experiments?

When designing Western blot experiments with SPBC8E4.01c antibody, implement the following controls:

• Positive control: Include lysate from wild-type S. pombe cells grown in phosphate-limited conditions to upregulate the transporter

• Negative control: Use one of the following:

  • Lysate from an SPBC8E4.01c deletion strain

  • Lysate from a distantly related yeast species lacking SPBC8E4.01c homologs

  • Pre-incubation of the antibody with purified antigen (peptide competition)

• Loading control: Include an antibody against a constitutively expressed protein (e.g., actin or tubulin)

Additionally, calculate the expected molecular weight of SPBC8E4.01c based on the amino acid sequence and consider potential post-translational modifications that might affect migration patterns.

What is the optimal protocol for fission yeast lysate preparation to ensure SPBC8E4.01c detection?

For optimal detection of SPBC8E4.01c in fission yeast lysates, follow this specialized protocol:

• Cultivate S. pombe cells to mid-exponential phase (OD595 = 0.4-0.6, approximately 0.8-1.2 × 10^7 cells/ml)

• Harvest cells by centrifugation at 3,000g for 5 minutes at 4°C

• Wash cell pellet twice with cold PBS

• Resuspend cells in lysis buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 5 mM EDTA

  • Protease inhibitor cocktail

  • Phosphatase inhibitors (if studying phosphorylation)

• Add acid-washed glass beads (0.5 mm diameter) to the cell suspension

• Disrupt cells using a bead beater (8 cycles of 30 seconds on/30 seconds off on ice)

• Centrifuge at 14,000g for 15 minutes at 4°C to remove cell debris

• Transfer supernatant to a fresh tube and determine protein concentration

This method preserves membrane proteins like SPBC8E4.01c that can be challenging to extract and maintain in their native state.

What dilution range is recommended for SPBC8E4.01c antibody in Western blot applications?

For Western blot applications with SPBC8E4.01c polyclonal antibody, start with a 1:1000 dilution in 5% non-fat milk or BSA in TBST and adjust based on signal intensity. The following optimization table provides guidelines for troubleshooting:

Begin with overnight incubation at 4°C with gentle rocking, followed by 3-5 washes with TBST (5 minutes each). Optimize blocking conditions (BSA vs. milk) if high background persists. Remember that membrane proteins often require special handling to prevent aggregation during sample preparation.

How can I validate SPBC8E4.01c antibody specificity using yeast biopanning approaches?

To rigorously validate SPBC8E4.01c antibody specificity using yeast biopanning approaches, implement this advanced protocol:

• Prepare bait peptides:

  • Synthesize biotinylated peptides corresponding to the SPBC8E4.01c epitope recognized by the antibody

  • Include control peptides with single amino acid substitutions at critical positions

• Cell surface immobilization:

  • Culture HEK293FT cells to 80-90% confluence in 6-well plates

  • Dilute biotinylated peptides to 0.1 μM in PBSCM-BSA (total volume 1 mL per well)

  • Incubate cells with streptavidin followed by biotinylated peptides

• Antibody screening:

  • Prepare yeast cells expressing an scFv library at 10-fold excess of library size

  • Incubate yeast cells with immobilized peptides

  • Perform sequential rounds of selection with increasing stringency

  • Isolate and sequence yeast displaying high-affinity binders

• Validation in 96-well format:

  • Clone isolated binders into bi-directional yeast surface display plasmids expressing fluorescent proteins

  • Compare binding to target peptide versus control peptides

  • Perform whole-well image analysis to quantify specificity

This approach provides quantitative data on antibody specificity and cross-reactivity, enabling confident interpretation of experimental results.

What approaches can be used to study post-translational modifications of SPBC8E4.01c protein?

Investigating post-translational modifications (PTMs) of SPBC8E4.01c requires a multi-faceted approach:

• Phosphorylation analysis:

  • Generate phospho-specific antibodies against predicted phosphorylation sites

  • Validate using the yeast biopanning protocol described earlier

  • Perform immunoprecipitation followed by mass spectrometry

  • Compare phosphorylation patterns in cells grown under different phosphate concentrations

• Ubiquitination detection:

  • Express tagged ubiquitin (His6-Ub or HA-Ub) in S. pombe

  • Immunoprecipitate SPBC8E4.01c using the specific antibody

  • Blot for ubiquitin to detect modification

  • Alternatively, perform tandem affinity purification of both ubiquitin and SPBC8E4.01c

• Glycosylation assessment:

  • Treat cell lysates with deglycosylating enzymes (PNGase F, Endo H)

  • Compare migration patterns by Western blot before and after treatment

  • Perform lectin blotting to identify specific glycan structures

• Membrane localization studies:

  • Perform subcellular fractionation to isolate different membrane compartments

  • Use density gradient centrifugation to separate distinct vesicle populations

  • Quantify SPBC8E4.01c distribution across fractions by Western blot

These approaches provide comprehensive insights into the regulatory mechanisms governing SPBC8E4.01c function and trafficking.

How can I use SPBC8E4.01c antibody to study phosphate transport mechanisms in fission yeast?

To investigate phosphate transport using SPBC8E4.01c antibody, implement these advanced experimental strategies:

• Correlate protein expression with transport activity:

  • Culture cells under varying phosphate concentrations (0.1, 1, 5, 10 mM)

  • Measure uptake of radiolabeled phosphate (³²P) at each concentration

  • Quantify SPBC8E4.01c protein levels by Western blot

  • Plot transport activity versus protein expression to establish relationship

• Investigate protein-protein interactions:

  • Perform co-immunoprecipitation with SPBC8E4.01c antibody

  • Analyze precipitated complexes by mass spectrometry

  • Validate key interactions using reciprocal co-IP or proximity ligation assay

  • Map functional domains using truncation mutants

• Localization studies:

  • Use immunofluorescence to track SPBC8E4.01c localization under different conditions

  • Correlate localization with transport activity during cell cycle progression

  • Perform time-lapse imaging after phosphate re-addition to phosphate-starved cells

• Structure-function analysis:

  • Generate point mutations in predicted functional domains

  • Assess protein expression levels by Western blot

  • Correlate mutations with transport activity and cellular localization

These approaches provide mechanistic insights into phosphate transport regulation and the specific role of SPBC8E4.01c in this process.

How should I interpret non-specific bands when using SPBC8E4.01c antibody in Western blots?

When encountering non-specific bands with SPBC8E4.01c antibody, use this systematic approach for interpretation:

• Characterize the pattern:

  • Document molecular weights of all observed bands

  • Compare with predicted size of SPBC8E4.01c (accounting for PTMs)

  • Determine if additional bands are consistently observed across experiments

• Validate specificity:

  • Perform peptide competition assay (pre-incubate antibody with immunizing peptide)

  • Compare with SPBC8E4.01c deletion strain lysate

  • Test antibody on closely related species to assess cross-reactivity

• Investigate biological significance:

  • Determine if additional bands represent:

    • Degradation products (appear with increasing sample age)

    • Splice variants (consistent molecular weights)

    • Post-translationally modified forms (altered by specific treatments)

    • Protein complexes (disrupted by stronger denaturing conditions)

• Optimization strategies:

  • Adjust antibody dilution (try more dilute solutions)

  • Modify blocking conditions (switch between BSA and milk)

  • Increase wash stringency (higher salt concentration or longer washes)

  • Use gradient gels to improve separation around the expected molecular weight

Remember that polyclonal antibodies contain multiple antibody species recognizing different epitopes, which may contribute to detection of related proteins or modified forms.

What is the recommended protocol for validating SPBC8E4.01c antibody specificity in knockout strains?

To rigorously validate SPBC8E4.01c antibody specificity using knockout strains, follow this protocol:

• Generate knockout strain:

  • Use PCR-based gene targeting to replace SPBC8E4.01c with a selection marker

  • Confirm deletion by PCR and sequencing of the integration junctions

  • Verify phenotype if SPBC8E4.01c is non-essential or use diploid strains if essential

• Sample preparation:

  • Culture wild-type and knockout strains under identical conditions

  • Prepare lysates using the optimized protocol described in FAQ 2.2

  • Normalize protein loading by total protein measurement

  • Include phosphate starvation conditions to induce maximal expression in wild-type

• Western blot analysis:

  • Run samples from both strains in adjacent lanes

  • Include molecular weight markers spanning the expected protein size

  • Perform transfer and antibody incubation using standardized conditions

  • Include a control antibody against an unrelated protein to confirm equal loading

• Quantitative analysis:

  • Perform densitometry on bands at the expected molecular weight

  • Calculate signal-to-noise ratio for wild-type versus knockout samples

  • Determine statistical significance across biological replicates (n≥3)

A highly specific antibody should show robust signal in wild-type samples and complete absence of the specific band in knockout samples. Any residual bands in knockout samples represent non-specific binding.

What methods can be used to optimize SPBC8E4.01c antibody for immunofluorescence in fission yeast?

Optimizing SPBC8E4.01c antibody for immunofluorescence in fission yeast requires addressing the unique challenges of yeast cell wall and membrane protein detection:

• Cell wall digestion optimization:

  • Test various cell wall digestion enzymes:

    • Novozyme 234 (5 mg/ml in buffer A)

    • Zymolyase (1-5 mg/ml)

    • Glusulase (5 μl/ml)

  • Optimize digestion time (30-90 minutes) and temperature (25-37°C)

  • Monitor protoplast formation microscopically (cells become round when cell wall is removed)

• Fixation method comparison:

  • Test multiple fixation methods in parallel:

    • 4% paraformaldehyde (10-20 minutes)

    • Methanol fixation (-20°C, 6 minutes)

    • Combined formaldehyde/methanol fixation

  • Assess each method for structural preservation and epitope accessibility

• Permeabilization optimization:

  • Compare detergents for membrane permeabilization:

    • 0.1% Triton X-100

    • 0.1-0.5% NP-40

    • 0.1% Saponin (may better preserve membrane proteins)

  • Optimize incubation time (5-15 minutes)

• Signal amplification strategies:

  • Implement tyramide signal amplification for weak signals

  • Use fluorophore-conjugated secondary antibodies with brightness matched to signal strength

  • Consider biotin-streptavidin amplification systems

• Controls and imaging parameters:

  • Include SPBC8E4.01c deletion strain as negative control

  • Co-stain with markers of relevant cellular compartments

  • Optimize exposure settings to prevent saturation

  • Perform z-stack imaging to capture the entire cell volume

This systematic approach addresses the key challenges in visualizing membrane transporters like SPBC8E4.01c in the complex cellular architecture of fission yeast.

How can I quantitatively analyze SPBC8E4.01c expression levels across different growth conditions?

For rigorous quantitative analysis of SPBC8E4.01c expression levels:

• Experimental design for comparable data:

  • Culture S. pombe under standardized conditions (temperature, media composition)

  • Sample cells at matched growth phases (mid-exponential, OD595 = 0.4-0.6)

  • Include biological replicates (n≥3) for statistical analysis

  • Process all samples in parallel to minimize technical variations

• Quantitative Western blot protocol:

  • Use a standard curve of recombinant SPBC8E4.01c or serially diluted reference sample

  • Apply samples in technical triplicates

  • Include multiple loading controls (actin, GAPDH, total protein stain)

  • Capture images within linear dynamic range of detection system

• Normalization and analysis:

  • Normalize SPBC8E4.01c signal to appropriate loading control

  • Calculate fold change relative to control condition

  • Perform statistical analysis (ANOVA with post-hoc tests for multiple conditions)

  • Present data with error bars representing standard deviation or standard error

• Validation with orthogonal methods:

  • Confirm protein changes with mRNA quantification (RT-qPCR)

  • Consider absolute quantification using mass spectrometry

  • Correlate expression with functional assays (phosphate uptake)

This methodical approach enables reliable comparison of SPBC8E4.01c levels across diverse experimental conditions while minimizing technical artifacts.

What are the challenges in studying protein-protein interactions involving SPBC8E4.01c and how can they be addressed?

Studying protein-protein interactions of membrane transporters like SPBC8E4.01c presents several challenges that require specialized approaches:

• Challenges and solutions in co-immunoprecipitation:

  • Challenge: Membrane protein solubilization

  • Solution: Test multiple detergents (DDM, digitonin, CHAPS) at various concentrations; use crosslinking agents like DSP or formaldehyde prior to cell lysis

• Challenges in preserving transient interactions:

  • Challenge: Capturing dynamic phosphate transport-related interactions

  • Solution: Implement in vivo proximity labeling using BioID or APEX2 fused to SPBC8E4.01c; compare interactomes under phosphate-replete and phosphate-limited conditions

• Challenges in distinguishing direct from indirect interactions:

  • Challenge: Co-IP captures entire complexes

  • Solution: Perform in vitro binding assays with purified components; use yeast two-hybrid with membrane yeast two-hybrid (MYTH) system specifically designed for membrane proteins

• Challenges in detecting low-abundance interactors:

  • Challenge: Important regulatory proteins may be present at low levels

  • Solution: Implement stable isotope labeling (SILAC) followed by mass spectrometry; use multiple negative controls to filter out common contaminants

• Challenges in confirming biological relevance:

  • Challenge: Distinguishing meaningful interactions from artifacts

  • Solution: Validate key interactions using multiple methods; demonstrate functional consequences of disrupting interactions through targeted mutations

This systematic approach addresses the specific challenges of studying membrane protein interactions while providing multiple layers of validation.

How can I design experiments to investigate the regulation of SPBC8E4.01c in response to phosphate availability?

To comprehensively investigate SPBC8E4.01c regulation in response to phosphate availability:

• Transcriptional regulation analysis:

  • Culture cells in media with varying phosphate concentrations (0, 0.1, 1, 10 mM)

  • Measure SPBC8E4.01c mRNA levels by RT-qPCR

  • Perform chromatin immunoprecipitation to identify transcription factors binding to the promoter

  • Use reporter constructs with mutated promoter elements to map regulatory regions

• Post-translational regulation characterization:

  • Monitor protein levels by Western blot across phosphate concentrations

  • Assess protein half-life using cycloheximide chase experiments

  • Identify phosphorylation sites by immunoprecipitation followed by mass spectrometry

  • Generate phospho-mimetic and phospho-null mutants to assess functional impact

• Subcellular localization dynamics:

  • Track SPBC8E4.01c localization using immunofluorescence or fluorescent protein tagging

  • Perform time-lapse imaging after phosphate addition or removal

  • Quantify protein distribution between plasma membrane and internal compartments

  • Co-localize with markers of trafficking pathways (early endosomes, recycling endosomes)

• Correlation with transport activity:

  • Measure ³²P uptake rates under matching conditions

  • Calculate transport kinetics parameters (Km, Vmax)

  • Correlate transport activity with protein levels and localization

  • Assess impact of regulatory mutants on transport function

This multi-level analysis provides a comprehensive understanding of how SPBC8E4.01c responds to environmental phosphate availability at transcriptional, post-translational, trafficking, and functional levels.

How can I apply advanced screening techniques to identify novel regulators of SPBC8E4.01c function?

To identify novel regulators of SPBC8E4.01c function using advanced screening approaches:

• Genome-wide deletion/mutation library screening:

  • Express epitope-tagged SPBC8E4.01c in S. pombe deletion library

  • Screen for mutations affecting SPBC8E4.01c localization, stability, or phosphorylation

  • Use high-content microscopy for phenotypic analysis

  • Validate hits with secondary assays measuring phosphate transport activity

• CRISPR-based screens:

  • Implement CRISPR interference (CRISPRi) or activation (CRISPRa) systems in S. pombe

  • Target transcription factors, kinases, and trafficking components

  • Monitor SPBC8E4.01c expression and localization

  • Perform screens under both phosphate-replete and phosphate-limited conditions

• Synthetic genetic array analysis:

  • Cross SPBC8E4.01c mutant (hypomorphic allele) with genome-wide deletion collection

  • Identify synthetic lethal/sick interactions

  • Map genetic interaction network to identify functional pathways

  • Validate key interactions with targeted experiments

• Proteomic approaches:

  • Perform temporal analysis of SPBC8E4.01c phosphorylation during phosphate starvation

  • Identify differentially regulated sites by quantitative phosphoproteomics

  • Map kinase-substrate relationships using selective inhibitors

  • Create a dynamic model of SPBC8E4.01c regulation

These advanced screening approaches generate unbiased, systems-level insights into SPBC8E4.01c regulation that may reveal unexpected regulatory mechanisms and potential targets for manipulation of phosphate transport.

What techniques can I use to correlate SPBC8E4.01c function with cellular phosphate homeostasis in real-time?

To correlate SPBC8E4.01c function with phosphate homeostasis in real-time:

• Genetically encoded phosphate sensors:

  • Express fluorescent phosphate sensors (e.g., cpFLIPPi) in S. pombe

  • Perform ratiometric imaging to measure cytosolic phosphate concentrations

  • Compare dynamics in wild-type versus SPBC8E4.01c mutant cells

  • Track rapid changes in phosphate levels following environmental perturbations

• Multimodal imaging approaches:

  • Combine fluorescently tagged SPBC8E4.01c with phosphate sensors

  • Perform simultaneous imaging of protein localization and phosphate levels

  • Use microfluidics for precise control of extracellular environment

  • Quantify temporal relationships between transporter redistribution and phosphate uptake

• Single-cell analysis techniques:

  • Implement flow cytometry with phosphate-responsive reporters

  • Sort cells based on phosphate levels or SPBC8E4.01c expression

  • Analyze population heterogeneity in response to phosphate stress

  • Correlate cellular phosphate content with growth rate or cell cycle progression

• Complementary biochemical measurements:

  • Develop methods for rapid fractionation of cell populations at defined timepoints

  • Measure compartment-specific phosphate concentrations

  • Track phosphate distribution between cytosol, vacuole, and other organelles

  • Correlate compartmentalization with SPBC8E4.01c activity