SPAC1039.04 Antibody

What is SPAC1039.04 and why is it significant in fission yeast research?

SPAC1039.04 refers to a specific gene in Schizosaccharomyces pombe (fission yeast, strain 972/ATCC 24843) encoding a protein that has become an important target for cellular biology research. Antibodies against this protein are essential tools for investigating protein expression, localization, and function in yeast models. The significance lies in its utility for studying fundamental cellular processes in this model organism that can be extrapolated to understand conserved mechanisms across eukaryotes .

What are the key specifications of commercially available SPAC1039.04 antibodies?

The commercially available SPAC1039.04 antibody is a rabbit polyclonal antibody purified by antigen affinity chromatography. It is raised against recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) SPAC1039.04 protein. The antibody is unconjugated (not linked to enzymes or fluorophores) and has been validated for ELISA and Western blot applications. The product typically includes 200μg of antigens (used as positive control) and 1ml pre-immune serum (used as negative control) alongside the purified rabbit polyclonal antibodies .

How does SPAC1039.04 antibody compare with other yeast protein detection systems?

Unlike monoclonal antibodies that target a single epitope, the polyclonal nature of SPAC1039.04 antibody provides recognition of multiple epitopes on the target protein, potentially offering enhanced sensitivity for detection in various applications. This characteristic makes it particularly valuable for research applications where protein conformation may be altered during experimental procedures. While some antibodies in yeast research require species-specific optimizations, SPAC1039.04 antibody has been specifically developed for recognition of S. pombe proteins, reducing cross-reactivity concerns that might be encountered with less specific detection methods .

What are the validated applications for SPAC1039.04 antibody?

The SPAC1039.04 antibody has been validated for two primary applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Suitable for quantitative determination of SPAC1039.04 protein levels in solution.

• Western Blot (WB): Effective for detecting denatured SPAC1039.04 protein from cell or tissue lysates separated by gel electrophoresis.

The antibody has been specifically tested against yeast species reactivity, making it appropriate for Schizosaccharomyces pombe research applications .

What is the recommended methodology for Western blot using SPAC1039.04 antibody?

When performing Western blot with SPAC1039.04 antibody, researchers should follow this methodological approach:

• Sample Preparation: Extract proteins from S. pombe using appropriate lysis buffers containing protease inhibitors

• Protein Separation: Separate proteins via SDS-PAGE (typically 10-12% gels)

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane

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

• Primary Antibody Incubation: Dilute SPAC1039.04 antibody (recommended starting dilution 1:1000) in blocking buffer and incubate overnight at 4°C

• Washing: Wash 3-5 times with TBST

• Secondary Antibody: Incubate with anti-rabbit HRP-conjugated secondary antibody

• Detection: Develop using ECL substrate and appropriate imaging system

Include both positive control (provided antigen) and negative control (pre-immune serum) to validate specificity of detection .

How should researchers optimize ELISA protocols for SPAC1039.04 detection?

For ELISA optimization with SPAC1039.04 antibody, consider this methodological approach:

• Plate Coating: Coat microplate wells with purified SPAC1039.04 protein or sample containing the target

• Blocking: Block with 1-5% BSA in PBS

• Antibody Titration: Perform an antibody titration (starting from 1:500 to 1:5000) to determine optimal concentration

• Incubation Parameters: Test different incubation times (1-4 hours) and temperatures (room temperature vs. 4°C)

• Detection System: Use HRP-conjugated secondary antibody and appropriate substrate

• Controls: Include the provided positive control antigen and pre-immune serum as negative control

• Standard Curve: Establish a standard curve using the provided antigen at known concentrations

The FDA guidelines on immunoassay development emphasize minimizing wash steps for detecting both low and high-affinity antibodies in screening assays, which can be applied when optimizing SPAC1039.04 antibody ELISA protocols .

How can researchers validate the specificity of SPAC1039.04 antibody reactions?

To validate antibody specificity, implement these methodological approaches:

• Positive and Negative Controls: Use the provided antigen (200μg) as a positive control and pre-immune serum (1ml) as a negative control in all experiments

• Knockout/Knockdown Validation: Test the antibody in SPAC1039.04 knockout or knockdown S. pombe strains, where a true specific antibody should show reduced or absent signal

• Competitive Binding Assay: Pre-incubate the antibody with purified SPAC1039.04 protein before application in your detection system; specific binding should be blocked

• Cross-Reactivity Assessment: Test against related yeast proteins to ensure specificity

• Multiple Detection Methods: Confirm results using complementary techniques (e.g., if detected by Western blot, confirm with immunofluorescence)

This comprehensive validation approach follows principles similar to those used in therapeutic antibody development, where confirmatory assays demonstrate antibody specificity for target proteins .

What controls should be incorporated when using SPAC1039.04 antibody?

A robust experimental design should include these controls:

• Positive Control:

  • Use the provided antigen (200μg) to verify antibody activity

  • Include wild-type S. pombe extracts with known SPAC1039.04 expression

• Negative Controls:

  • Pre-immune serum (1ml provided with the antibody) to establish background levels

  • SPAC1039.04 knockout/knockdown samples if available

  • Secondary antibody-only control to check for non-specific binding

• Loading Controls:

  • For Western blots, include detection of housekeeping proteins (e.g., actin, tubulin)

  • For immunofluorescence, include nuclear or membrane markers

• Dilution Series:

  • Test a range of antibody dilutions to determine optimal signal-to-noise ratio

Implementing these controls ensures experimental rigor and follows principles similar to those established for clinical antibody testing .

How can researchers address weak or absent signals when using SPAC1039.04 antibody?

When encountering weak or absent signals, consider these methodological interventions:

• Antibody Concentration: Increase antibody concentration gradually (e.g., from 1:1000 to 1:500 or 1:250)

• Protein Extraction Method: Optimize lysis conditions to ensure complete protein extraction and preservation of epitopes

  • Test different lysis buffers (RIPA, NP-40, Triton X-100)

  • Include appropriate protease inhibitors

• Detection Enhancement:

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

  • Use more sensitive detection systems (enhanced chemiluminescence substrates)

  • Consider signal amplification methods

• Protein Expression Verification:

  • Confirm SPAC1039.04 expression in your samples via RT-PCR

  • Consider whether experimental conditions might alter expression levels

• Antibody Storage and Handling:

  • Ensure proper storage at -20°C or -80°C

  • Avoid multiple freeze-thaw cycles

  • Prepare fresh working dilutions for each experiment

These approaches are consistent with general principles of antibody-based detection optimization while being specifically tailored to yeast protein detection challenges .

What are common sources of background and non-specific binding with SPAC1039.04 antibody?

When troubleshooting high background or non-specific binding, consider these methodological solutions:

• Blocking Optimization:

  • Test different blocking agents (BSA, non-fat dry milk, normal serum)

  • Increase blocking time or concentration

• Washing Protocol Enhancement:

  • Increase number of washes

  • Add detergent (0.05-0.1% Tween-20) to wash buffers

  • Extend washing times

• Antibody Dilution:

  • Further dilute primary and secondary antibodies

  • Pre-absorb antibody with yeast extract from species other than S. pombe

• Sample Preparation Issues:

  • Ensure complete lysis and removal of cellular debris

  • Centrifuge lysates thoroughly before loading

• Cross-Reactivity Management:

  • Test for cross-reactivity with related proteins

  • Consider using more stringent washing conditions

This troubleshooting approach is consistent with best practices in immunoassay development while addressing specific challenges encountered in yeast protein detection .

How can SPAC1039.04 antibody be adapted for multi-parameter analyses?

For advanced multi-parameter analyses, researchers can implement these methodological approaches:

• Multiplexed Immunofluorescence:

  • Combine SPAC1039.04 antibody with antibodies against other proteins of interest

  • Use secondary antibodies with distinct fluorophores

  • Implement spectral unmixing for optimal signal separation

• Co-Immunoprecipitation (Co-IP) Applications:

  • Utilize SPAC1039.04 antibody to pull down protein complexes

  • Identify interaction partners via mass spectrometry

  • Validate interactions with reciprocal Co-IPs

• ChIP-Seq Integration:

  • If SPAC1039.04 has DNA-binding capabilities, adapt the antibody for chromatin immunoprecipitation

  • Combine with next-generation sequencing for genome-wide binding profiles

• Single-Cell Analysis:

  • Optimize for flow cytometry applications

  • Consider mass cytometry (CyTOF) for high-dimensional analysis

• Proximity Ligation Assays:

  • Detect protein-protein interactions in situ

  • Combine with other detection methods for functional validation

These advanced applications follow similar methodological principles to those used in therapeutic antibody characterization and multi-parameter immunoassays .

What considerations should be made when comparing different lots of SPAC1039.04 antibody?

When working with different antibody lots, implement these methodological approaches for consistency:

• Lot-to-Lot Validation:

  • Test new lots in parallel with previous lots

  • Create a standardized positive control sample to benchmark performance

  • Document detection sensitivity and specificity metrics

• Calibration Curve Standardization:

  • Establish standard curves using provided antigens

  • Compare EC50 values and detection limits between lots

• Critical Epitope Assessment:

  • If available, perform epitope mapping to ensure consistent epitope recognition

  • Evaluate binding affinity using techniques like surface plasmon resonance

• Application-Specific Validation:

  • Re-validate each new lot for all applications (Western blot, ELISA, etc.)

  • Adjust protocols as needed based on lot-specific performance

• Documentation and Traceability:

  • Maintain detailed records of lot-specific performance

  • Include lot information in all experimental documentation

These approaches mirror the rigorous validation processes used in therapeutic antibody development and manufacturing, where lot-to-lot consistency is critical .

How can researchers effectively use the provided positive and negative controls?

The SPAC1039.04 antibody kit includes 200μg of antigens (positive control) and 1ml pre-immune serum (negative control). Here's how to leverage these controls effectively:

Positive Control Applications:

• Standard Curve Generation:

  • Create serial dilutions (2-fold or 3-fold) for quantitative assays

  • Establish detection limits and linear range

• Antibody Validation:

  • Confirm antibody activity before each experimental series

  • Use as reference point for expected signal intensity

• Troubleshooting Tool:

  • When experiments fail, test with positive control to distinguish between antibody issues and sample problems

Negative Control Applications:

• Background Determination:

  • Establish signal threshold for positive detection

  • Subtract background signal from experimental values

• Non-specific Binding Assessment:

  • Identify potential cross-reactivity issues

  • Optimize blocking and washing conditions

• Signal-to-Noise Optimization:

  • Calculate signal-to-noise ratios under different conditions

  • Refine protocol to maximize this ratio

This methodological approach aligns with FDA guidance on immunoassay development, which emphasizes the importance of proper controls for establishing assay specificity .

How should results be quantified and statistically analyzed?

For rigorous quantification and statistical analysis of SPAC1039.04 antibody results:

• Quantification Methods:

  Application	Quantification Method	Software Tools	Normalization Approach
  Western Blot	Densitometry	ImageJ, Image Lab	Normalize to loading control
  ELISA	Standard curve fitting	GraphPad Prism, R	Four-parameter logistic regression
  Immunofluorescence	Mean fluorescence intensity	ImageJ, CellProfiler	Background subtraction

• Statistical Analysis Approaches:

  • For comparison between experimental groups, use appropriate statistical tests (t-test, ANOVA)

  • Include minimum of three biological replicates for statistical power

  • Report both mean and standard deviation/standard error

  • Consider non-parametric tests if data doesn't follow normal distribution

• Data Visualization Best Practices:

  • Show representative images alongside quantification

  • Include error bars in all graphical representations

  • Consider dot plots rather than bar graphs to show data distribution

• Reproducibility Assessment:

  • Calculate intra-assay and inter-assay coefficients of variation

  • Define acceptance criteria before experiments (e.g., CV < 15%)

This analytical framework follows principles similar to those used in clinical antibody research and development programs .

What are the current limitations of SPAC1039.04 antibody research?

Understanding the limitations of SPAC1039.04 antibody research is critical for proper experimental design and data interpretation:

• Technical Limitations:

  • Being a polyclonal antibody, lot-to-lot variation may affect reproducibility

  • The antibody has been validated only for ELISA and Western blot applications

  • Specificity across different strains of S. pombe may vary

• Biological Context Limitations:

  • Expression levels of SPAC1039.04 under different growth conditions or stress responses are not well characterized

  • Potential post-translational modifications might affect epitope recognition

  • Protein interactions may mask antibody binding sites in certain experimental contexts

• Research Gap Limitations:

  • Limited published literature on SPAC1039.04 function and regulation

  • Incomplete characterization of cross-reactivity with closely related proteins

  • Absence of structural data on antibody-epitope interactions

• Methodological Alternatives to Consider:

  • Epitope tagging approaches (HA, FLAG, etc.) when antibody limitations are significant

  • CRISPR-mediated endogenous tagging for live-cell imaging applications

  • Mass spectrometry-based approaches for absolute quantification

Understanding these limitations helps researchers develop appropriate controls and alternative approaches when necessary .

How might advanced antibody engineering improve SPAC1039.04 detection?

Future advancements in antibody engineering could significantly enhance SPAC1039.04 detection through:

• Monoclonal Antibody Development:

  • Generation of monoclonal antibodies for improved lot-to-lot consistency

  • Epitope-specific antibodies for detecting different protein domains or forms

• Recombinant Antibody Technologies:

  • Development of single-chain variable fragments (scFvs) for improved tissue penetration

  • Creation of bispecific antibodies for simultaneous detection of SPAC1039.04 and interacting partners

• Affinity Maturation Approaches:

  • Engineering higher-affinity variants for enhanced sensitivity

  • Developing pH-dependent binding antibodies for improved signal-to-noise ratios

• Direct Conjugation Strategies:

  • Site-specific conjugation of fluorophores or enzymes to minimize functional interference

  • Novel conjugation chemistries for improved stability and sensitivity

These approaches mirror the advanced antibody engineering technologies employed in therapeutic antibody development, such as those used for the Abs-9 antibody against SpA5, which demonstrated nanomolar affinity and strong prophylactic efficacy .

What emerging technologies might complement SPAC1039.04 antibody-based detection?

Emerging technologies that could complement traditional antibody-based detection include:

• Single-Cell Sequencing Integration:

  • Combining antibody detection with single-cell RNA sequencing for correlating protein levels with transcriptomes

  • Similar to approaches used in identifying SpA5 antibodies through high-throughput single-cell RNA and VDJ sequencing

• Proximity-Based Detection Methods:

  • Proximity ligation assays for detecting protein-protein interactions in situ

  • BioID or APEX2 proximity labeling for identifying interaction networks

• Advanced Imaging Techniques:

  • Super-resolution microscopy for precise subcellular localization

  • Expansion microscopy for improved spatial resolution in complex samples

• AI-Enhanced Image Analysis:

  • Machine learning algorithms for automated detection and quantification

  • Deep learning approaches for identifying subtle phenotypic changes

• In Silico Modeling:

  • Structure prediction and molecular docking methods similar to those used with Abs-9 antibody

  • Epitope prediction algorithms for improving antibody design

These complementary approaches could significantly extend the utility and applications of SPAC1039.04 antibody in research settings.