SPAPB1A10.05 Antibody

What is SPAPB1A10.05 protein and what is its significance in yeast biology?

SPAPB1A10.05 (also known as VSP15a) is a 32 kDa protein found in yeast with Gene ID 2543600 and GenBank Accession Number CAC21478 . While the exact functional role of this protein remains under investigation, it serves as an important marker in yeast cellular studies. Current research utilizes antibodies against this protein primarily for detection and quantification in experimental systems.

To properly study this protein, researchers should:

• Compare sequence homology with related proteins using bioinformatics tools

• Investigate its expression patterns under different growth conditions

• Consider its potential interactions with other yeast proteins

• Examine evolutionary conservation across different yeast species

What applications has the VSP15a/SPAPB1A10.05 antibody been validated for?

According to the product information, the VSP15a antibody has been specifically validated for:

The VSP15a antibody is generated using a VSP15a fusion protein (Ag0724) as the immunogen . This makes it particularly suitable for studies requiring:

• Detection of native SPAPB1A10.05 protein in yeast samples

• Quantitative analysis of protein expression levels

• Validation of gene knockout or overexpression experiments

• Comparative studies across yeast strains

It's important to note that while ELISA is the primary validated application, methodical validation would be required before using this antibody for other applications such as Western blotting or immunohistochemistry .

How should samples be prepared for optimal detection with SPAPB1A10.05 antibody?

For maximizing detection sensitivity and specificity with SPAPB1A10.05 antibody, sample preparation should follow these methodological guidelines:

• Cell Harvesting and Processing:

  • Collect exponentially growing yeast cultures by centrifugation (3000-5000 × g for 5 minutes)

  • Wash cell pellets with cold PBS to remove media components

  • Lyse cells using an appropriate method:

    • Mechanical disruption with glass beads or sonication

    • Enzymatic treatment with zymolyase followed by gentle lysis

    • Chemical lysis with detergent-containing buffers

• Lysis Buffer Composition:

  • Base buffer: PBS or Tris-buffered solution (pH 7.3-7.5)

  • Protease inhibitors: Complete protease inhibitor cocktail

  • Detergents: 0.1-1% NP-40, Triton X-100, or RIPA buffer components

  • Preservatives: Consider adding 0.02% sodium azide for longer-term storage

• Post-lysis Processing:

  • Centrifuge lysates (10,000-20,000 × g for 10-15 minutes) to remove debris

  • Collect and quantify protein concentration in the supernatant

  • Normalize all samples to equal protein concentration

  • Aliquot to avoid repeated freeze-thaw cycles

• Storage and Handling:

  • Store samples at -80°C for long-term storage

  • For short-term use, keep at 4°C with protease inhibitors

  • Avoid repeated freeze-thaw cycles that might denature the target protein

This systematic approach ensures consistent and reliable detection while preserving the native state of the SPAPB1A10.05 protein for antibody recognition .

What controls should be included when using SPAPB1A10.05 antibody in experimental settings?

A robust experimental design with appropriate controls is essential when working with SPAPB1A10.05 antibody to ensure result validity and interpretability:

• Positive Controls:

  • Wild-type yeast strains known to express SPAPB1A10.05

  • Recombinant SPAPB1A10.05 protein (if available)

  • Previously validated yeast samples with confirmed antibody reactivity

• Negative Controls:

  • SPAPB1A10.05 knockout or deletion strains (if available)

  • Primary antibody omission control (to assess secondary antibody specificity)

  • Isotype control using normal rabbit IgG at equivalent concentration

  • Non-expressing yeast species or strains

• Specificity Controls:

  • Pre-absorption with immunizing antigen

  • Competitive binding with purified antigen

  • Samples with graded levels of target expression

• Technical Controls:

  • Loading controls for normalization

  • Serial dilution of samples to confirm linear detection range

  • Replicate samples to assess reproducibility

• Cross-validation:

  • When feasible, confirm key findings using an orthogonal detection method

  • Compare results to published literature on SPAPB1A10.05

What are common issues encountered with SPAPB1A10.05 antibody in ELISA, and how can they be resolved?

When using SPAPB1A10.05 antibody in ELISA applications, researchers may encounter several technical challenges. Here are evidence-based solutions for common issues:

• High Background Signal:

  • Problem: Non-specific binding causing elevated baseline readings

  • Solutions:

    • Optimize blocking (test 1-5% BSA, casein, or non-fat milk)

    • Increase washing steps (5-7 washes instead of standard 3)

    • Include 0.05-0.1% Tween-20 in wash and antibody dilution buffers

    • Pre-absorb antibody with non-target yeast lysates

    • Dilute primary antibody further (perform titration)

• Weak or No Signal:

  • Problem: Insufficient target detection despite confirmed presence

  • Solutions:

    • Verify sample preparation adequately exposes epitopes

    • Increase antibody concentration or incubation time

    • Optimize sample loading (concentration and volume)

    • Ensure antibody hasn't degraded during storage

    • Consider more sensitive detection systems

• Poor Reproducibility:

  • Problem: High variability between replicates or experiments

  • Solutions:

    • Standardize all protocols with detailed SOPs

    • Maintain consistent incubation times and temperatures

    • Prepare larger batches of working solutions

    • Include internal controls and standard curves

    • Normalize data to consistent reference samples

• Cross-Reactivity:

  • Problem: Detection of unintended targets

  • Solutions:

    • Validate specificity using genetic controls

    • Perform competitive binding assays

    • Use more stringent washing conditions

    • Pre-clear samples if necessary

Systematic troubleshooting by changing one parameter at a time will help isolate and resolve specific issues affecting experimental outcomes .

How can the specificity of SPAPB1A10.05 antibody be rigorously validated?

Rigorous validation of SPAPB1A10.05 antibody specificity is crucial for ensuring reliable experimental results. A comprehensive validation strategy should include:

• Genetic Validation Approaches:

  • Compare signals between wild-type and SPAPB1A10.05 knockout samples

  • Use RNA interference to create targeted knockdowns

  • Test samples with varying expression levels of the target protein

  • Correlate antibody signal with mRNA expression data

• Biochemical Validation Methods:

  • Western blot analysis to confirm detection of a single band at 32 kDa

  • Immunoprecipitation followed by mass spectrometry identification

  • Peptide competition assays using the immunizing antigen

  • Pre-absorption tests to eliminate specific binding

• Orthogonal Validation Techniques:

  • Compare results with alternative detection methods

  • Use an independent antibody targeting a different epitope

  • Employ recombinant expression systems with tagged proteins

  • Cross-validate findings using proteomics approaches

• Specificity Assessments:

  • Test for cross-reactivity with related yeast proteins

  • Evaluate performance across different sample types

  • Examine reactivity in different yeast species or strains

  • Analyze sequence homology of potential cross-reactive proteins

• Quantitative Validation Parameters:

  • Establish detection limits and linear range

  • Determine reproducibility across multiple experimental runs

  • Document lot-to-lot consistency when using commercial antibodies

This multi-faceted validation approach provides strong evidence for antibody specificity and increases confidence in experimental results .

How can SPAPB1A10.05 antibody be integrated with other techniques to study protein interactions?

Integrating SPAPB1A10.05 antibody with complementary techniques creates powerful experimental platforms for studying protein interactions in yeast systems:

• Immunoprecipitation-Based Approaches:

  • Co-immunoprecipitation (Co-IP) to pull down SPAPB1A10.05 and associated proteins

  • IP followed by mass spectrometry for unbiased interactome analysis

  • Chromatin immunoprecipitation (ChIP) if SPAPB1A10.05 has DNA-binding properties

  • Reciprocal Co-IP with antibodies against suspected interaction partners

• Microscopy-Based Integration:

  • Immunofluorescence with SPAPB1A10.05 antibody for localization studies

  • Dual-labeling with potential interaction partners

  • Proximity ligation assay (PLA) for direct visualization of protein-protein interactions

  • Super-resolution microscopy for detailed spatial relationship analysis

• Functional Analysis Integration:

  • Antibody-mediated inhibition studies to assess functional consequences

  • Domain mapping using deletion mutants and antibody recognition

  • Kinetic studies tracking SPAPB1A10.05 interactions during cellular processes

  • Correlation of interaction data with phenotypic outcomes

• Validation Through Orthogonal Methods:

  • Confirm antibody-based findings using yeast two-hybrid systems

  • Validate interactions with recombinant protein binding assays

  • Cross-reference with published interactome databases

  • Compare with computational predictions of protein-protein interactions

This integrated approach provides multiple lines of evidence for protein interactions, strengthening confidence in the biological significance of findings and revealing functional relationships within broader cellular networks .

How can epitope mapping be performed to identify the specific region of SPAPB1A10.05 recognized by the antibody?

Epitope mapping of the SPAPB1A10.05 antibody provides critical information about antibody specificity and potential cross-reactivity. A comprehensive epitope mapping strategy includes:

• Peptide Array Analysis:

  • Generate overlapping peptides (15-20 amino acids with 5 amino acid overlap) spanning the SPAPB1A10.05 sequence

  • Immobilize peptides on a membrane or microarray format

  • Probe with the SPAPB1A10.05 antibody and appropriate secondary detection

  • Identify reactive peptides representing the linear epitope components

  • Narrow down the epitope by testing shorter peptides within reactive regions

• Deletion/Truncation Analysis:

  • Create a series of N-terminal and C-terminal truncation constructs

  • Express truncated proteins in a suitable system

  • Test antibody binding via Western blot or ELISA

  • Progressively narrow down the region containing the epitope

• Site-Directed Mutagenesis:

  • Once a candidate region is identified, perform alanine scanning mutagenesis

  • Substitute individual amino acids to identify critical residues for binding

  • Express these mutants and quantify changes in antibody binding affinity

• Structural Approaches:

  • Use hydrogen-deuterium exchange mass spectrometry to identify protected regions

  • If resources permit, obtain structural data through X-ray crystallography or cryo-EM

  • Apply computational modeling to predict antibody-antigen interactions

The epitope mapping data from these approaches can be used to:

• Predict potential cross-reactivity with homologous proteins

• Design blocking peptides for competition assays

• Understand the structural basis of antibody specificity

• Guide development of improved antibodies or detection reagents

How does the SPAPB1A10.05 antibody compare to other yeast protein antibodies in research applications?

When comparing SPAPB1A10.05 antibody to other yeast protein antibodies, several factors should be considered for appropriate research application selection:

When selecting between SPAPB1A10.05 antibody and alternatives, researchers should carefully match antibody characteristics to their specific experimental requirements, considering both technical specifications and biological questions being addressed .

What emerging research approaches might benefit from SPAPB1A10.05 antibody application?

Several emerging research approaches could benefit from incorporating SPAPB1A10.05 antibody in experimental workflows:

• High-Throughput Proteomics:

  • Integration into automated ELISA platforms for large-scale screening

  • Validation of mass spectrometry-based protein identification

  • Targeted protein quantification in complex samples

  • Confirmation of protein expression patterns across multiple conditions

• Single-Cell Analysis:

  • Development of microfluidic antibody-based detection systems

  • Validation of single-cell proteomics findings

  • Analysis of cell-to-cell variability in protein expression

  • Correlation of SPAPB1A10.05 levels with phenotypic heterogeneity

• Synthetic Biology Applications:

  • Monitoring protein expression in engineered yeast strains

  • Quality control for yeast-based bioproduction systems

  • Validation of genetic circuit function in synthetic biology

  • Assessment of protein production stability over time

• Comparative Yeast Biology:

  • Investigation of orthologs across different yeast species

  • Analysis of protein conservation in evolutionary studies

  • Examination of expression in non-conventional yeasts

  • Correlation of protein presence with phenotypic variations

• Advanced Imaging Technologies:

  • Adaptation for super-resolution microscopy applications

  • Development of proximity labeling approaches

  • Integration with emerging spatial proteomics methods

  • Correlation with live-cell imaging data

These emerging approaches represent opportunities to extend the utility of SPAPB1A10.05 antibody beyond traditional applications, potentially yielding new insights into yeast biology and broader applications in biotechnology and comparative genomics .

What are the key considerations for using SPAPB1A10.05 antibody in cross-species studies?

When applying SPAPB1A10.05 antibody in cross-species research contexts, several methodological considerations are essential:

• Epitope Conservation Analysis:

  • Perform sequence alignment of the target region across species of interest

  • Quantify amino acid identity and similarity percentages

  • Predict epitope accessibility in homologous proteins using structural models

  • Consider evolutionary distance between target species and original yeast antigen

• Validation Requirements:

  • Test antibody reactivity with purified proteins from each target species

  • Perform Western blot analysis on lysates from multiple species

  • Include appropriate positive controls (yeast samples) and negative controls

  • Validate any cross-species signals using genetic approaches when possible

• Protocol Optimization:

  • Adjust antibody concentration for each species through titration experiments

  • Modify sample preparation to account for species-specific differences

  • Optimize buffer conditions and incubation parameters for each system

  • Consider species-specific blocking reagents to minimize background

• Interpretation Considerations:

  • Be cautious with negative results (may indicate lack of cross-reactivity)

  • Consider evolutionary relationships when comparing signal intensity

  • Account for potential differences in protein abundance or modifications

  • Acknowledge limitations of cross-species applications in research reports

• Complementary Approaches:

  • Use multiple detection methods to corroborate findings

  • Consider generating species-specific antibodies for critical applications

  • Employ recombinant expression systems for controlled comparisons

  • Utilize genomic data to support interpretation of antibody results

These methodological considerations can help researchers appropriately apply and interpret results when extending SPAPB1A10.05 antibody use beyond its primary yeast target system .

How should researchers approach the development of assays using SPAPB1A10.05 antibody for specialized applications?

Developing specialized assays with SPAPB1A10.05 antibody requires a systematic approach to ensure reliability, sensitivity, and specificity:

• Assay Design Framework:

  • Define clear research objectives and required assay parameters

  • Select appropriate assay format (direct, indirect, sandwich ELISA, etc.)

  • Consider sample type and anticipated concentration range

  • Design appropriate positive and negative controls

• Antibody Characterization:

  • Determine antibody affinity and specificity parameters

  • Assess concentration-dependent performance characteristics

  • Evaluate pH, temperature, and buffer stability

  • Test compatibility with assay components and detection systems

• Methodological Optimization:

  • Systematically vary key parameters:

    • Antibody concentration (titration experiments)

    • Sample volume and dilution

    • Incubation time and temperature

    • Buffer composition and pH

    • Blocking reagent type and concentration

    • Washing stringency and duration

• Assay Validation:

  • Determine analytical sensitivity (limit of detection)

  • Establish analytical specificity (cross-reactivity profile)

  • Assess precision (repeatability and reproducibility)

  • Evaluate accuracy (spike recovery experiments)

  • Test robustness to minor protocol variations

• Protocol Documentation:

  • Create detailed standard operating procedures

  • Document all optimization experiments and results

  • Establish quality control criteria and acceptance parameters

  • Include troubleshooting guidelines for common issues

For specialized applications like high-throughput screening, automated platforms, or diagnostic development, additional considerations might include:

• Adaptation to microplate or microfluidic formats

• Stability of reagents during extended processing

• Compatibility with automation equipment

• Statistical approach for data analysis and interpretation

This structured development approach maximizes the likelihood of creating a reliable, fit-for-purpose assay using SPAPB1A10.05 antibody for specialized research applications .