SPAC4G8.08 Antibody

What is SPAC4G8.08 and why is it of interest to researchers?

SPAC4G8.08 is a gene in Schizosaccharomyces pombe (fission yeast) that encodes a putative mitochondrial iron ion transporter. It is also referred to as mrs3+ in some research contexts. This protein is of interest to researchers studying mitochondrial function, iron transport, and cellular metabolism in yeast models. According to genomic screens, the SPAC4G8.08 gene deletion mutant shows altered sensitivity to antifungal agents, particularly becoming resistant to terbinafine but not clotrimazole, suggesting its involvement in membrane integrity or drug response pathways .

What are the basic molecular characteristics of the SPAC4G8.08 protein?

The SPAC4G8.08 protein (UniProt ID: Q09834) is characterized as a putative mitochondrial iron ion transporter. The encoding gene has been sequenced and contains an open reading frame (ORF) of 816 base pairs. Research data indicates the protein is localized to mitochondria and is involved in iron ion transport, which is crucial for various cellular processes including respiration and enzyme function .

How are antibodies against yeast proteins like SPAC4G8.08 typically generated?

Antibodies against yeast proteins such as SPAC4G8.08 are typically generated through several approaches:

• Recombinant protein expression: The target gene is cloned into an expression vector (like pcDNA3.1+/C-(K)DYK), expressed in bacteria (E. coli), and the purified protein is used as an immunogen .

• Synthetic peptide approach: Short, unique peptide sequences from the target protein are synthesized and conjugated to carrier proteins before immunization.

• Genetic immunization: DNA encoding the target protein is directly injected into animals to induce antibody production.

For SPAC4G8.08 specifically, researchers typically use affinity purification techniques with the recombinant protein approach. The immunization process often involves rabbits for polyclonal antibodies or mice for monoclonal antibody development, with subsequent screening for specificity against the target protein .

What methods should I use to validate an antibody against SPAC4G8.08?

Validation of antibodies against yeast proteins like SPAC4G8.08 is critical for research reliability. A comprehensive validation approach should include:

• Genetic validation: Testing the antibody in wild-type vs. knockout/deletion strains is the gold standard method. This approach demonstrates if the antibody can distinguish between the presence and absence of the target protein .

• Western blot analysis: Comparing protein expression in SPAC4G8.08 overexpression strains versus deletion mutants to confirm specificity.

• Immunoprecipitation (IP): Performing IP followed by mass spectrometry to confirm the identity of the precipitated protein.

• Cross-reactivity testing: Ensuring the antibody doesn't recognize related proteins, which is particularly important for mitochondrial transporter family members.

• Cell fractionation: Confirming localization to mitochondrial fractions as expected for a mitochondrial iron transporter.

Research has shown that genetic validation approaches (using knockout strains) significantly outperform orthogonal validation approaches in confirming antibody specificity. A study of 614 commercial antibodies found that 89% of antibodies validated using genetic approaches could detect their intended target in Western blot applications, compared to 80% of those validated using orthogonal approaches .

How can I address potential cross-reactivity issues with antibodies against yeast proteins?

Cross-reactivity is a significant concern with antibodies against yeast proteins due to homology between related protein families. To address this issue:

• Pre-adsorption controls: Incubate the antibody with recombinant SPAC4G8.08 protein before immunostaining to block specific binding sites.

• Test in multiple strains: Evaluate antibody performance across various yeast strains with different expression levels of SPAC4G8.08.

• Competitive binding assays: Use increasing concentrations of purified antigen to demonstrate specific displacement of antibody binding.

• Epitope mapping: Identify the specific region of SPAC4G8.08 recognized by the antibody and perform sequence analysis to identify potential cross-reactive proteins.

• Secondary antibody selection: Use highly cross-adsorbed secondary antibodies like Donkey Anti-Goat IgG(H+L) with minimal reactivity to other species when performing multi-protein detection experiments .

What are the optimal experimental conditions for Western blot detection of SPAC4G8.08?

Based on research protocols for similar yeast mitochondrial proteins, the following conditions are recommended for Western blot detection of SPAC4G8.08:

• Sample preparation:

  • Perform spheroplasting of S. pombe cells as described in protocols to enhance protein extraction

  • Use a lysis buffer containing 1% NP-40, protease inhibitor cocktail, and phosphatase inhibitors

  • For mitochondrial proteins, include digitonin (0.5-1%) in buffer to solubilize membrane proteins

• Gel and transfer conditions:

  • Use 10-12% SDS-PAGE gels for proper separation

  • Transfer to PVDF membrane at 100V for 1 hour or 30V overnight at 4°C

• Antibody incubation:

  • Primary antibody dilution: 1:500 to 1:1000 (to be optimized)

  • Incubation: Overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-species antibody at 1:5000 dilution for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) detection

  • Include known positive controls and SPAC4G8.08 deletion mutant as a negative control

Based on research with similar proteins, the expected molecular weight would be approximately the predicted size from the amino acid sequence, potentially with post-translational modifications affecting migration pattern .

How can I optimize immunoprecipitation protocols for SPAC4G8.08?

Immunoprecipitation (IP) of yeast mitochondrial proteins like SPAC4G8.08 requires specific considerations:

• Cell preparation:

  • For surface protein biotinylation, treat live cells with sulfo-NHS-biotin prior to lysis

  • Use gentle lysis conditions with 1% NP-40 or digitonin to preserve protein conformation

• IP procedure:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Use 2-5 μg of antibody per 500 μg of total protein

  • Include appropriate controls: IgG control and ideally a SPAC4G8.08 deletion strain

• Washing and elution:

  • Use stringent washing to remove non-specific interactions

  • Elute with acidic glycine buffer or SDS-PAGE sample buffer depending on downstream applications

• Verification:

  • Confirm IP success by Western blot or mass spectrometry

  • Use streptavidin-HRP probing if biotinylation was performed, as demonstrated in the 214D8 antibody characterization study

What are effective methods for immunolocalization of SPAC4G8.08 in yeast cells?

Immunolocalization of mitochondrial proteins in yeast requires specialized techniques:

• Cell preparation:

  • Fix cells with 3.7% formaldehyde for 30 minutes

  • Digest cell wall with zymolyase to create spheroplasts

  • Perform gentle permeabilization with 0.1% Triton X-100

• Immunostaining:

  • Block with 1% BSA and 0.1% gelatin

  • Use primary antibody at 1:50 to 1:200 dilution

  • Apply fluorophore-conjugated secondary antibodies at 1:500 dilution

  • Co-stain with mitochondrial markers (MitoTracker) for colocalization

• Controls and verification:

  • Include peptide competition controls

  • Use SPAC4G8.08 deletion strains as negative controls

  • Compare with known mitochondrial markers

• Imaging:

  • Use confocal microscopy with appropriate filter sets

  • Perform z-stack imaging to capture the 3D distribution of signals

  • Quantify colocalization with mitochondrial markers using ImageJ or similar software

How can I use SPAC4G8.08 antibody to study drug responses in fission yeast?

SPAC4G8.08 (mrs3+) has been implicated in antifungal drug responses in S. pombe. To study this relationship:

• Drug sensitivity profiling:

  • Compare wild-type and SPAC4G8.08 mutant strains using standardized assays (streak, spot, and microtiter assays)

  • Assess growth in the presence of terbinafine and clotrimazole at various concentrations

  • Quantify drug sensitivity using methods described in genomic screen studies

• Protein expression analysis:

  • Use Western blotting with anti-SPAC4G8.08 antibody to measure protein expression changes in response to drug treatment

  • Compare expression levels across different time points of drug exposure

• Protein-protein interactions:

  • Perform IP with anti-SPAC4G8.08 antibody before and after drug treatment

  • Use mass spectrometry to identify interacting partners that may change with drug exposure

• Localization changes:

  • Conduct immunofluorescence studies to detect any redistribution of SPAC4G8.08 in response to drug treatment

  • Correlate localization changes with cellular phenotypes

Research data indicates that SPAC4G8.08 deletion mutants show resistance to terbinafine, suggesting its involvement in drug susceptibility mechanisms. This can be further investigated by combining antibody-based approaches with functional assays .

What techniques can be used to study SPAC4G8.08 interactions with other mitochondrial proteins?

To investigate protein-protein interactions involving SPAC4G8.08:

• Co-immunoprecipitation (Co-IP):

  • Use anti-SPAC4G8.08 antibody to pull down the protein complex

  • Analyze co-precipitated proteins by mass spectrometry

  • Confirm specific interactions by reverse Co-IP with antibodies against identified partners

• Proximity labeling techniques:

  • Create SPAC4G8.08 fusion with biotin ligase (BioID) or APEX2

  • Express in yeast cells and activate labeling

  • Purify biotinylated proteins and identify by mass spectrometry

• Crosslinking mass spectrometry:

  • Apply protein crosslinkers to intact yeast cells or isolated mitochondria

  • Immunoprecipitate SPAC4G8.08 under denaturing conditions

  • Analyze crosslinked peptides by mass spectrometry to map interaction interfaces

• Split reporter assays:

  • Create fusions of SPAC4G8.08 and candidate interactors with split GFP or split ubiquitin

  • Monitor reconstitution of the reporter as evidence of protein-protein interaction

Data from BioGRID indicates that SPAC4G8.08 has at least 6 identified interactors in S. pombe, which can be further characterized using these approaches .

How can active learning strategies improve antibody validation for proteins like SPAC4G8.08?

Recent research on antibody validation demonstrates that active learning approaches can significantly enhance validation efficiency:

• Sequential testing strategy:

  • Begin with small-scale validation using knockout controls

  • Apply machine learning to predict which additional tests would provide the most information

  • Iteratively expand validation based on predicted outcomes

• Library-on-library screening:

  • Test antibody binding against arrays of related proteins

  • Apply active learning algorithms to identify optimal test conditions

  • Recent studies show this approach can reduce the number of required test variants by up to 35%

• Multi-application validation:

  • Rather than validating for a single application, use a matrix approach

  • Test antibody in Western blot, immunoprecipitation, and immunofluorescence concurrently

  • Use machine learning to identify patterns predictive of successful application

• Computational epitope prediction:

  • Apply AlphaFold2 and molecular docking methods to predict antibody-antigen interactions

  • Use these predictions to design targeted validation experiments

  • This approach has been successful in antibody development against pathogens like Staphylococcus aureus

When applied to yeast proteins like SPAC4G8.08, these approaches could significantly reduce the resources needed for comprehensive validation while increasing confidence in results.

Why might I observe multiple bands when detecting SPAC4G8.08 by Western blot?

Multiple bands in Western blot detection of SPAC4G8.08 could result from several factors:

• Post-translational modifications:

  • Phosphorylation or other modifications can cause mobility shifts

  • Different mitochondrial targeting stages (precursor vs. mature form)

  • Test with phosphatase treatment before SDS-PAGE to determine if phosphorylation contributes

• Proteolytic processing:

  • Mitochondrial proteins often undergo processing after import

  • N-terminal targeting sequences may be cleaved

  • Use N- and C-terminal targeted antibodies to distinguish fragments

• Cross-reactivity:

  • Related mitochondrial transporters may share epitopes

  • Test specificity using SPAC4G8.08 deletion strains

  • Perform peptide competition assays to confirm specific binding

• Technical issues:

  • Insufficient sample denaturation can cause aggregation

  • Increase SDS concentration or boiling time

  • Try different sample preparation methods for membrane proteins, such as inclusion of urea or guanidine hydrochloride

To distinguish between these possibilities, use SPAC4G8.08 deletion mutants as negative controls and complement with epitope-tagged versions of the protein for comparison.

What considerations are important when choosing secondary antibodies for detection of SPAC4G8.08?

Selecting appropriate secondary antibodies is crucial for specific detection:

• Cross-adsorption requirements:

  • Choose secondary antibodies that are cross-adsorbed against other species present in your experiment

  • For yeast studies, secondary antibodies cross-adsorbed against human, mouse, rat, and rabbit proteins minimize background

  • Products like Donkey Anti-Goat IgG(H+L) with multi-species cross-adsorption offer high specificity

• Detection system compatibility:

  • HRP-conjugated secondaries work well for chemiluminescent detection

  • Fluorophore-conjugated secondaries allow multiplexing and quantification

  • Biotinylated secondaries provide amplification options for low-abundance proteins

• Isotype specificity:

  • Match secondary antibody to the isotype of your primary antibody

  • For IgG3 kappa primary antibodies, use secondaries specific to this isotype

  • Consider using anti-light chain specific secondaries when detecting immunoprecipitated proteins

• Optimization parameters:

  • Titrate secondary antibody concentration (typically 1:2000-1:10000)

  • Optimize incubation time and temperature

  • Include appropriate washing steps to reduce background

Data from antibody validation studies show that proper secondary antibody selection can significantly impact detection sensitivity and specificity, particularly for low-abundance proteins like mitochondrial transporters .

How can I improve protein extraction efficiency for mitochondrial membrane proteins like SPAC4G8.08?

Extracting membrane-associated mitochondrial proteins presents unique challenges:

• Cell disruption methods:

  • For yeast cells, use mechanical disruption (glass beads) or enzymatic methods (zymolyase)

  • Spheroplasting with zymolyase before gentle lysis can improve protein extraction

  • Optimize lysis conditions to prevent protein degradation

• Buffer composition:

  • Include specialized detergents: digitonin (0.5-2%), DDM (1%), or Triton X-100 (1%)

  • Add protease inhibitor cocktails specifically designed for yeast

  • Include reducing agents like DTT (1-5 mM) to prevent oxidation

• Subcellular fractionation:

  • Isolate mitochondria before protein extraction to enrich for SPAC4G8.08

  • Use differential centrifugation or density gradient methods

  • Verify mitochondrial fractions using markers like cytochrome c oxidase

• Sample preparation for SDS-PAGE:

  • Heat samples at moderate temperature (37°C) rather than boiling to prevent aggregation

  • Include urea (2-4 M) in sample buffer for improved solubilization

  • Consider specialized gel systems for membrane proteins