YLL058W Antibody

What is YLL058W and why is it significant for yeast research?

YLL058W is a previously uncharacterized gene in Saccharomyces cerevisiae (Baker's yeast) that encodes an alternative homocysteine synthase. Its significance lies in challenging the long-held assumption that Met15 was the only enzyme capable of inorganic sulfur assimilation in yeast. Recent research has demonstrated that YLL058W enables cells lacking Met15 to assimilate enough inorganic sulfur for survival and proliferation, although these cells require exogenous methionine or other organosulfurs for optimal growth in patches or liquid cultures . This discovery has profound implications for the hundreds of experiments that have used MET15 as a genetic marker in foundational studies of eukaryote genetics and systems biology.

What are the key properties of the YLL058W antibody?

The YLL058W antibody is a rabbit-derived polyclonal antibody that specifically targets the YLL058W protein from Saccharomyces cerevisiae (strain ATCC 204508 / S288c). It is supplied in liquid form containing 0.03% Proclin 300 preservative, 50% glycerol, and 0.01M PBS at pH 7.4. The antibody has been purified using antigen affinity methods and is IgG isotype. Upon receipt, it should be stored at -20°C or -80°C, with repeated freeze-thaw cycles avoided to maintain efficacy . This antibody has been validated for ELISA and Western blot applications specifically for research purposes.

What experimental applications has the YLL058W antibody been tested for?

The YLL058W antibody has been validated specifically for ELISA (Enzyme-Linked Immunosorbent Assay) and Western blot (WB) applications . These techniques enable researchers to detect and quantify YLL058W protein expression in yeast samples and investigate its regulation under various experimental conditions. When using these methods, proper identification of the antigen should be ensured through appropriate controls.

How should YLL058W antibody be stored and handled for optimal performance?

For optimal antibody performance, YLL058W antibody should be stored at either -20°C or -80°C immediately upon receipt . To preserve antibody activity, it is advisable to aliquot the stock solution into smaller volumes to minimize freeze-thaw cycles, which can degrade antibody quality. When working with the antibody, allow it to thaw completely at 4°C before use, and briefly centrifuge to collect the solution at the bottom of the tube. All antibody dilutions should be prepared fresh in appropriate buffers, and working solutions should be kept cold during experiments to maintain binding specificity.

How should I design Western blot experiments using YLL058W antibody?

When designing Western blot experiments with YLL058W antibody, consider the following protocol:

• Sample preparation:

  • Extract proteins from yeast cells using mechanical disruption (glass beads) or enzymatic methods

  • Include protease inhibitors to prevent degradation of target proteins

  • Quantify total protein concentration using Bradford or BCA assays

• Gel electrophoresis and transfer:

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

  • Transfer proteins to PVDF or nitrocellulose membranes at 100V for 1 hour or 30V overnight

• Antibody incubation:

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

  • Incubate with YLL058W antibody (1:1000 dilution as starting point) overnight at 4°C

  • Wash 3× with TBST for 5 minutes each

  • Incubate with HRP-conjugated anti-rabbit secondary antibody for 1 hour

  • Wash 3× with TBST for 5 minutes each

• Controls:

  • Include wild-type yeast lysate as positive control

  • Include YLL058W knockout lysate as negative control if available

  • Use housekeeping protein detection (e.g., actin) for loading control

What are the optimal conditions for ELISA using YLL058W antibody?

For optimal ELISA performance with YLL058W antibody, follow these guidelines:

• Plate coating:

  • Coat high-binding 96-well plates with purified antigen or yeast lysate in carbonate buffer (pH 9.6)

  • Incubate overnight at 4°C

  • Wash 3× with PBS containing 0.05% Tween-20 (PBST)

• Blocking and antibody incubation:

  • Block with 3% BSA in PBST for 2 hours at room temperature

  • Add YLL058W antibody (begin with 1:1000 dilution) in 1% BSA-PBST

  • Incubate for 2 hours at room temperature or overnight at 4°C

  • Wash 5× with PBST

• Detection:

  • Add HRP-conjugated anti-rabbit secondary antibody (1:5000) in 1% BSA-PBST

  • Incubate for 1 hour at room temperature

  • Wash 5× with PBST

  • Develop with TMB substrate and read absorbance at 450nm after stopping with 2N H₂SO₄

What controls should be included when using YLL058W antibody?

To ensure experimental validity, include the following controls:

Including these controls allows for proper interpretation of results and troubleshooting if unexpected results occur.

How can I use YLL058W antibody to study the relationship between YLL058W and Met15 in sulfur metabolism?

To investigate the functional relationship between YLL058W and Met15 in sulfur metabolism, design experiments that:

• Compare protein expression levels under different sulfur conditions:

  • Use Western blotting with YLL058W antibody to quantify expression in wild-type, met15Δ, and yll058wΔ strains grown in media with varying sulfur sources (inorganic sulfate, methionine, cysteine)

  • Monitor temporal expression changes during adaptation to sulfur limitation

• Perform co-immunoprecipitation studies:

  • Use YLL058W antibody to pull down protein complexes

  • Analyze interacting partners by mass spectrometry to identify potential regulatory proteins or metabolic enzymes

• Conduct genetic interaction studies:

  • Create double mutants (met15Δ yll058wΔ) and analyze phenotypes

  • Use YLL058W antibody to monitor compensatory changes in related proteins

• Investigate subcellular localization:

  • Develop immunofluorescence protocols using YLL058W antibody

  • Compare localization patterns under different sulfur conditions

This approach will help elucidate how these two pathways complement each other and respond to environmental changes.

How can I validate the specificity of my YLL058W antibody results?

To validate YLL058W antibody specificity, employ multiple complementary approaches:

• Genetic validation:

  • Compare antibody signal in wild-type vs. yll058wΔ strains

  • Use strains expressing epitope-tagged YLL058W and detect with both YLL058W antibody and tag-specific antibody

• Biochemical validation:

  • Perform peptide competition assays by pre-incubating antibody with immunizing peptide

  • Confirm protein identity by immunoprecipitation followed by mass spectrometry

• Methodological validation:

  • Test multiple antibody dilutions to establish optimal signal-to-noise ratio

  • Compare results across different detection methods (chemiluminescence, fluorescence)

  • Prepare samples using alternative lysis methods to ensure consistent detection

These validation steps ensure that experimental observations truly reflect YLL058W biology rather than artifacts or cross-reactivity.

How can I investigate post-translational modifications of YLL058W protein?

Post-translational modifications of YLL058W can be studied using the antibody in conjunction with specialized techniques:

• Phosphorylation analysis:

  • Treat lysates with phosphatase before Western blotting

  • Use Phos-tag gels to separate phosphorylated forms

  • Perform immunoprecipitation with YLL058W antibody followed by phospho-specific staining or mass spectrometry

• Other modifications:

  • Use specific inhibitors of post-translational modifications (deacetylase inhibitors, proteasome inhibitors)

  • Perform 2D gel electrophoresis to separate modified forms before Western blotting

  • Combine with specific antibodies against modifications (ubiquitin, SUMO, methylation)

• Functional analysis:

  • Compare modification patterns under different growth conditions and stressors

  • Correlate modifications with enzyme activity assays

  • Engineer mutants at potential modification sites and analyze with YLL058W antibody

This approach can reveal regulatory mechanisms controlling YLL058W activity in response to cellular conditions.

What are common issues encountered when using YLL058W antibody and how can they be resolved?

When working with YLL058W antibody, researchers may encounter several common issues:

For persistent issues, consider antibody validation using recombinant YLL058W protein or comparative analysis with other detection methods.

How should I interpret conflicting data involving YLL058W expression or function?

When confronted with conflicting data regarding YLL058W:

• Examine methodological differences:

  • Different antibody lots or sources may have varying specificities

  • Sample preparation methods can affect protein detection

  • Growth conditions and media composition significantly impact expression

• Consider biological variables:

  • Genetic background effects (strain differences)

  • Growth phase and metabolic state variations

  • Environmental stressors affecting expression

• Validation approaches:

  • Use multiple independent methods to assess expression/function

  • Conduct genetic complementation studies

  • Perform dose-response experiments under controlled conditions

• Data integration:

  • Combine protein data with mRNA expression analysis

  • Correlate with metabolic intermediates of sulfur pathways

  • Consider evolutionary context when comparing between species

Careful consideration of these factors allows for proper interpretation of seemingly contradictory results.

How might YLL058W antibody be used in systems biology approaches?

YLL058W antibody can contribute to systems biology research through:

• Protein interaction networks:

  • Immunoprecipitation followed by mass spectrometry to identify interaction partners

  • Temporal analysis of interaction changes during sulfur limitation

  • Integration with genetic interaction data to build comprehensive networks

• Multi-omics integration:

  • Correlation of protein levels (detected by YLL058W antibody) with transcriptomics, metabolomics, and phenotypic data

  • Development of predictive models for sulfur metabolism incorporating YLL058W function

  • Characterization of regulatory networks controlling YLL058W expression

• Single-cell analysis:

  • Development of immunofluorescence protocols for studying cell-to-cell variation

  • Correlation with other markers to understand metabolic heterogeneity

  • Integration with microfluidic approaches for dynamic studies

These applications will advance our understanding of how alternative metabolic pathways contribute to cellular robustness and adaptation.

What is the evolutionary significance of YLL058W and how can this be studied?

The evolutionary significance of YLL058W can be investigated through:

• Comparative genomics:

  • Using YLL058W antibody to study protein expression in related yeast species

  • Correlation of expression patterns with ecological niches and metabolic capabilities

  • Analysis of selection pressure on YLL058W sequence versus Met15

• Functional conservation:

  • Cross-reactivity testing of YLL058W antibody with homologs in other species

  • Complementation studies using YLL058W orthologs from diverse fungi

  • Investigation of structural differences affecting catalytic efficiency

• Genomic context analysis:

  • Study of YLL058W's telomeric location and its implications for regulation

  • Analysis of chromosomal rearrangements affecting YLL058W across species

  • Investigation of horizontal gene transfer possibilities

This evolutionary perspective provides insight into how alternative metabolic pathways arise and persist through natural selection.