SPBPB10D8.06c Antibody

What is SPBPB10D8.06c and what is its functional significance in S. pombe?

SPBPB10D8.06c is a gene in the fission yeast Schizosaccharomyces pombe that encodes a membrane protein with putative sulfite transport functions. It belongs to a family of four tandem duplication genes (SPBPB10D8.04c, SPBPB10D8.05c, SPBPB10D8.06c, and SPBPB10D8.07c) that are homologous to the SSU1 gene encoding the sulfite efflux transporter in Saccharomyces cerevisiae . This protein is likely involved in sulfite metabolism, particularly in the transport of SO₃²⁻ (sulfite), which is an intermediate in the sulfur assimilation pathway.

The genomic context of SPBPB10D8.06c is noteworthy, as it is located within a heterochromatin domain known as "HOOD-16" on chromosome 2 . This chromosomal region (position 91,600-101,684) contains several membrane proteins and exhibits specific epigenetic marks that may regulate its expression under different growth conditions.

What are the key technical specifications of the SPBPB10D8.06c antibody?

The commercially available SPBPB10D8.06c antibody is supplied in liquid form with the following specifications:

When properly stored, the antibody maintains stability for at least 12 months from the date of receipt.

How can researchers validate the specificity of the SPBPB10D8.06c antibody?

Validation of antibody specificity is critical for ensuring experimental reliability. For SPBPB10D8.06c antibody, consider these methodological approaches:

• Knockout validation: Generate a SPBPB10D8.06c deletion strain using established genetic techniques for S. pombe. The antibody should show no signal in western blots or immunofluorescence when used on knockout cells.

• Epitope mapping: Perform targeted deletion or mutation of potential epitope regions in the SPBPB10D8.06c gene to identify the specific binding site.

• Cross-reactivity assessment: Test the antibody against closely related proteins, particularly SPBPB10D8.04c, SPBPB10D8.05c, and SPBPB10D8.07c, which share sequence similarity.

• Immunoprecipitation followed by mass spectrometry: This approach, similar to that described for antibody Abs-9 against SpA5 , can confirm that the antibody specifically pulls down SPBPB10D8.06c from yeast lysates.

• Recombinant protein expression: Express SPBPB10D8.06c with an orthogonal tag (e.g., His or GST) and verify that the antibody recognizes the pure recombinant protein.

What are the recommended protocols for western blotting with SPBPB10D8.06c antibody?

For optimal western blotting results with the SPBPB10D8.06c antibody, the following protocol is recommended based on established methods for S. pombe membrane proteins:

Cell Lysis and Protein Extraction:

• Harvest cells at mid-logarithmic phase (OD₆₀₀ = 0.5-0.8)

• Wash cells with ice-cold 10 mM NaPO₄, 0.5 mM EDTA, pH 7.5 buffer

• Lyse cells using glass beads in cold lysis buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 100 mM NaCl, and 0.5% NP-40 with protease inhibitor cocktail)

• Centrifuge at 13,000 rpm at 4°C for 5 minutes to clarify lysates

SDS-PAGE and Immunoblotting:

• Resolve proteins on 6-12% SDS-PAGE gels (10% recommended for SPBPB10D8.06c)

• Transfer to PVDF membrane at 100V for 1 hour or 30V overnight

• Block membrane in 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with SPBPB10D8.06c antibody (1:1000 dilution) overnight at 4°C

• Wash 3× with TBST, 10 minutes each

• Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour

• Wash 3× with TBST, 10 minutes each

• Develop using ECL system

For challenging membrane proteins like SPBPB10D8.06c, consider adding 8M urea to the sample buffer to improve denaturation before loading.

How can researchers effectively employ SPBPB10D8.06c antibody in immunofluorescence studies?

Immunofluorescence microscopy using SPBPB10D8.06c antibody requires careful sample preparation due to the yeast cell wall and the protein's membrane localization:

Optimized Immunofluorescence Protocol:

• Cell Wall Digestion and Fixation:

  • Fix cells with 3.7% formaldehyde for 30 minutes

  • Digest cell wall using zymolyase (1mg/ml) or lysing enzymes in sorbitol buffer

  • Permeabilize with 1% Triton X-100 for 5 minutes

• Immunolabeling Procedure:

  • Block with 1% BSA, 0.1% Tween-20 in PBS for 1 hour

  • Incubate with SPBPB10D8.06c antibody (1:100 dilution) overnight at 4°C

  • Wash 3× with PBS/0.1% Tween-20

  • Incubate with fluorophore-conjugated secondary antibody (1:200) for 1 hour

  • Counterstain with DAPI to visualize nuclei

  • Mount in anti-fade medium

This approach is similar to methods used for subcellular localization studies of Sup11p, another S. pombe protein involved in cell wall synthesis .

What considerations are important when using SPBPB10D8.06c antibody for co-localization studies?

When conducting co-localization studies with SPBPB10D8.06c antibody and other markers:

• Marker Selection:

  • Use established markers for specific cellular compartments:

    • Sec72-GFP for ER

    • Anp1-RFP for Golgi

    • FM4-64 for vacuolar/endosomal membranes

    • Calcofluor white for cell wall

• Fixation Compatibility:

  • Ensure fixation methods preserve all antigens of interest

  • For dual immunofluorescence, test antibodies individually first

• Sequential vs. Simultaneous Detection:

  • For antibodies from the same species, use sequential detection with blocking steps

  • For antibodies from different species, simultaneous incubation may be possible

• Microscopy Settings:

  • Use appropriate filter sets to avoid bleed-through

  • Perform controls with single antibodies to confirm specificity

  • Consider confocal microscopy for better resolution of membrane proteins

This approach has been effective for localizing other S. pombe membrane proteins such as Fft3, which influences nuclear organization and chromatin structure of insulators and subtelomeres .

How can SPBPB10D8.06c antibody be utilized to study the relationship between sulfite transport and cell wall integrity?

To investigate the functional relationship between sulfite transport and cell wall integrity using SPBPB10D8.06c antibody:

• Stress Response Studies:

  • Expose S. pombe cells to cell wall stressors (e.g., calcofluor white, Congo red)

  • Use the antibody to track changes in SPBPB10D8.06c expression and localization

  • Combine with transcript analysis (qRT-PCR) to correlate protein and mRNA levels

• Genetic Interaction Analysis:

  • Employ the antibody in studies of double mutants lacking SPBPB10D8.06c and cell wall integrity pathway components

  • Analyze the protein's expression in stress-activated MAPK pathway mutants (e.g., sty1Δ)

• Co-immunoprecipitation:

  • Use the antibody to identify interaction partners through co-IP followed by mass spectrometry

  • Focus on potential interactions with β-glucan synthesis regulators like Sup11p

• Correlative Microscopy:

  • Combine immunolocalization of SPBPB10D8.06c with cell wall component visualization using specific dyes

  • Track dynamic changes during cell cycle progression, particularly during septum formation

The relationship between cell wall remodeling and stress responses in S. pombe has been investigated for other proteins, such as the relationship between the stress-activated protein kinase (SAPK) pathway and cytokinesis .

What experimental approaches can be used to study SPBPB10D8.06c in the context of nuclear organization?

Based on findings that some membrane proteins in S. pombe influence nuclear organization , researchers can employ these approaches using SPBPB10D8.06c antibody:

• Chromatin Immunoprecipitation (ChIP):

  • Use the antibody to identify potential associations between SPBPB10D8.06c and chromatin regions

  • Follow protocols similar to those used for studying Fft3's role in controlling nuclear organization

• Fluorescence In Situ Hybridization (FISH) Combined with Immunofluorescence:

  • Combine FISH targeting specific genomic regions with immunolocalization of SPBPB10D8.06c

  • Examine whether SPBPB10D8.06c associates with particular chromosomal domains, especially HOOD-16

• Live-Cell Imaging with Complementary Techniques:

  • Use GFP-tagged nuclear envelope markers alongside immunostaining for SPBPB10D8.06c

  • Track dynamic changes in both signals during cell cycle progression

• Electron Microscopy with Immunogold Labeling:

  • Employ the antibody with gold particle conjugates for high-resolution localization

  • Examine potential associations with nuclear pores or specific nuclear envelope domains

This multi-faceted approach would build on observations that membrane proteins like Fft3 can mediate associations between specific chromatin domains and the nuclear envelope .

How can researchers apply active learning strategies to optimize SPBPB10D8.06c antibody-based assays?

Active learning approaches, similar to those used for antibody-antigen binding prediction , can be applied to optimize SPBPB10D8.06c antibody-based experimental designs:

• Iterative Assay Optimization:

  • Start with a small set of experimental conditions

  • Systematically expand testing based on initial results

  • Use machine learning to predict optimal conditions for subsequent iterations

• Epitope Refinement:

  • Apply computational prediction methods like those used in Alphafold2 and molecular docking

  • Validate predicted epitopes experimentally

  • Refine predictions based on experimental feedback

• Cross-Reactivity Analysis:

  • Test against recombinant fragments of related proteins (SPBPB10D8.04c, SPBPB10D8.05c, SPBPB10D8.07c)

  • Use data to train specificity prediction models

  • Apply insights to design more specific immunoassays

• Library-on-Library Screening:

  • Generate antigen variants through site-directed mutagenesis

  • Test antibody binding against the variant library

  • Apply machine learning to identify critical binding determinants

This approach can reduce the number of required experiments by up to 35% while accelerating optimization by approximately 28 steps compared to random testing .

What strategies are recommended for troubleshooting weak or absent signals when using SPBPB10D8.06c antibody?

When encountering weak or absent signals with SPBPB10D8.06c antibody, implement the following systematic troubleshooting approach:

• Sample Preparation Issues:

  • Ensure sufficient protein concentration (≥20 μg total protein per lane)

  • Test alternative lysis buffers optimized for membrane proteins

  • Consider using specialized extraction methods like spheroblasting

• Antibody-Related Factors:

  • Titrate antibody concentration (test 1:500, 1:1000, 1:2000 dilutions)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Test different lots of the antibody if available

• Protocol Optimization:

  Parameter	Standard Condition	Alternative Conditions to Test
  Blocking agent	5% milk	3% BSA, commercial blockers
  Incubation temperature	4°C overnight	Room temperature (4 hours)
  Membrane type	PVDF	Nitrocellulose
  Detection system	ECL	Enhanced ECL, fluorescent detection

• Expression Level Considerations:

  • Verify that experimental conditions support SPBPB10D8.06c expression

  • Consider that SPBPB10D8.06c may be expressed at low levels under standard conditions

  • Test induction with relevant stressors (e.g., sulfite exposure)

• Epitope Accessibility:

  • For immunofluorescence, test alternative fixation methods

  • For western blotting, ensure complete denaturation with 8M urea or heated SDS

These approaches address common issues encountered with antibodies targeting low-abundance membrane proteins in yeast.

How can researchers study SPBPB10D8.06c in relation to other members of the sulfite transporter family?

To investigate SPBPB10D8.06c in relation to other sulfite transporters (SPBPB10D8.04c, SPBPB10D8.05c, SPBPB10D8.07c):

• Comparative Expression Analysis:

  • Use the antibody in conjunction with mRNA analysis to determine relative expression levels

  • Compare protein and transcript levels under various sulfur-related stress conditions

  • Correlate expression with sulfite resistance phenotypes

• Functional Redundancy Studies:

  • Combine the antibody with genetic approaches using single and multiple knockouts

  • Quantify protein levels of remaining transporters when one or more are deleted

  • Test compensatory upregulation of remaining family members

• Domain-Specific Analysis:

  • Generate domain-specific antibodies or epitope tags

  • Compare localization patterns of different family members

  • Identify shared vs. unique interaction partners

• Evolutionary Context:

  • Compare SPBPB10D8.06c expression and function with homologs in related species

  • Correlate protein conservation with functional conservation

This approach builds on existing knowledge of the four tandem duplication genes and their putative roles in sulfite transport .

What methodological considerations are important when using SPBPB10D8.06c antibody for chromatin immunoprecipitation (ChIP)?

While SPBPB10D8.06c is primarily a membrane protein, some membrane proteins have been shown to associate with chromatin. If investigating potential chromatin associations:

• Cross-linking Optimization:

  • Test both formaldehyde (1-3%) and alternative cross-linkers (e.g., DSG)

  • Optimize cross-linking times (5-20 minutes) to capture transient interactions

  • Consider dual cross-linking approaches for membrane protein-DNA interactions

• Sonication Parameters:

  • Optimize sonication to yield DNA fragments of 200-500 bp

  • Verify fragment size by agarose gel electrophoresis

  • Consider alternative fragmentation methods (e.g., enzymatic digestion)

• Immunoprecipitation Conditions:

  • Test various antibody amounts (2-10 μg per reaction)

  • Optimize bead type (Protein A/G, magnetic vs. agarose)

  • Consider pre-clearing lysates to reduce background

• Controls and Validation:

  • Include non-specific IgG and input controls

  • Use knockout strains as negative controls

  • Validate by sequential ChIP with known interacting factors

• Data Analysis:

  • Compare enrichment to known heterochromatin domains like HOODs

  • Focus on regions associated with cell wall and sulfur metabolism genes

  • Correlate ChIP data with expression analysis

These considerations address the specific challenges of performing ChIP with antibodies targeting membrane proteins that may have chromatin associations, similar to approaches used for studying Fft3 and Dhp1 .

How can SPBPB10D8.06c antibody be integrated into multi-omics studies of sulfur metabolism in yeast?

To incorporate SPBPB10D8.06c antibody into comprehensive multi-omics studies:

• Integrated Experimental Design:

  • Combine antibody-based protein quantification with:

    • Transcriptomics (RNA-seq of sulfur metabolism genes)

    • Metabolomics (focusing on sulfur-containing metabolites)

    • Phenomics (growth assays under varying sulfur conditions)

• Time-Course Analysis:

  • Track SPBPB10D8.06c protein levels alongside transcriptional and metabolic changes

  • Monitor dynamic responses to sulfur limitation or toxicity

  • Correlate protein expression with metabolic flux changes

• Data Integration Framework:

  • Develop computational models that integrate protein expression data with other omics layers

  • Identify regulatory networks controlling SPBPB10D8.06c expression

  • Use machine learning to predict functional outcomes based on protein expression patterns

• Validation Approaches:

  • Use genetic perturbations (knockouts, overexpression) to validate model predictions

  • Apply CRISPR-based strategies to engineer precise modifications

  • Test model predictions under various environmental conditions

This integrative approach would build on existing knowledge of sulfur metabolism in S. pombe while providing new insights into the specific role of SPBPB10D8.06c.

What new research directions could emerge from applying monoclonal antibody development techniques to SPBPB10D8.06c?

Applying advanced monoclonal antibody development techniques to SPBPB10D8.06c could open new research avenues:

• Structure-Guided Epitope Selection:

  • Use structural prediction methods like AlphaFold2 to identify optimal epitopes

  • Design antibodies targeting functional domains of SPBPB10D8.06c

  • Apply approaches similar to those used for developing antibodies against infectious agents

• Conformation-Specific Antibodies:

  • Develop antibodies that specifically recognize active vs. inactive conformations

  • Create tools to monitor conformational changes during transport activity

  • Generate antibodies that distinguish between different post-translational modifications

• Cross-Species Functional Analysis:

  • Create antibodies that recognize conserved epitopes across multiple yeast species

  • Enable comparative studies of homologous transporters in pathogenic and non-pathogenic fungi

  • Investigate evolutionary conservation of sulfite transport mechanisms

• Biosensor Development:

  • Engineer antibody fragments for real-time monitoring of SPBPB10D8.06c dynamics

  • Develop FRET-based sensors to detect protein-protein interactions

  • Create tools for monitoring transport activity in living cells

These approaches could transform SPBPB10D8.06c from a relatively uncharacterized protein into a well-understood component of fungal sulfur metabolism, with potential applications in both basic science and biotechnology.

How might insights from SPBPB10D8.06c studies inform broader understanding of membrane protein biology?

Research on SPBPB10D8.06c using specialized antibodies can contribute to fundamental questions in membrane protein biology:

• Evolutionary Conservation of Transport Mechanisms:

  • Compare SPBPB10D8.06c structure and function with transporters across fungal species

  • Identify conserved regulatory mechanisms for sulfite transport

  • Trace the evolutionary history of this protein family through comparative studies

• Membrane Protein Quality Control:

  • Investigate how SPBPB10D8.06c is folded, targeted, and maintained in the membrane

  • Study protein degradation pathways for misfolded transporters

  • Examine stress-induced changes in protein turnover

• Membrane Domain Organization:

  • Explore potential association of SPBPB10D8.06c with specialized membrane domains

  • Investigate lipid requirements for proper function

  • Examine interactions with the cell wall synthesis machinery

• Transporter Regulation Paradigms:

  • Study how cellular sulfur status regulates transporter activity

  • Investigate post-translational modifications that control transport function

  • Develop models for coordinated regulation of multiple transporters