SPBC713.05 Antibody

What is SPBC713.05 and why is it studied in research?

SPBC713.05 is an uncharacterized WD repeat-containing protein found in Schizosaccharomyces pombe (fission yeast). It belongs to the WD repeat MORG1 family and is primarily localized in the cytoplasm and nucleus. As part of ongoing efforts to characterize the S. pombe proteome, studying this protein helps elucidate the function of WD repeat proteins in eukaryotic cellular processes. WD repeat proteins typically serve as platforms for protein-protein interactions and are involved in diverse cellular functions including signal transduction, cell cycle regulation, and vesicular trafficking.

What applications is the SPBC713.05 antibody suitable for?

The SPBC713.05 antibody has been tested and validated for several applications including:

• Western blotting (WB)

• Enzyme-linked immunosorbent assay (ELISA)

• Immunocytochemistry (ICC)

Different antibody formats may have varying performance in these applications. For example, polyclonal antibodies against SPBC713.05 are generally more sensitive for detection but may show lower specificity compared to monoclonal versions .

What are the subcellular localization patterns expected when using SPBC713.05 antibody?

When performing immunocytochemistry with SPBC713.05 antibody, researchers should expect to observe both cytoplasmic and nuclear localization patterns. This is consistent with the predicted subcellular distribution of the protein. When optimizing staining protocols, it is advisable to use cell fractionation controls to confirm the specificity of the observed localization pattern.

How should I validate the specificity of SPBC713.05 antibody before experimental use?

Comprehensive validation of SPBC713.05 antibody should follow multiple approaches based on the "five pillars" of antibody characterization :

• Genetic strategy validation: Test the antibody using wild-type S. pombe cells compared with SPBC713.05 knockout or knockdown strains. The absence of signal in genetic knockout models represents the gold standard for specificity confirmation.

• Orthogonal strategy validation: Compare protein expression results from antibody-based detection with antibody-independent methods (e.g., mass spectrometry or RNA-seq data).

• Independent antibody validation: Use multiple antibodies targeting different epitopes of SPBC713.05 and confirm consistent results.

• Recombinant expression validation: Overexpress tagged SPBC713.05 in cells and confirm detection of the overexpressed protein.

• Immunocapture MS validation: Use mass spectrometry to identify proteins captured by the SPBC713.05 antibody in immunoprecipitation experiments.

For maximum confidence, at least two of these approaches should be employed before using the antibody in critical experiments .

What controls should I include when using SPBC713.05 antibody in Western blot experiments?

To ensure reliable Western blot results with SPBC713.05 antibody, include these essential controls :

Additionally, running a dilution series of your sample can help establish the dynamic range and sensitivity of the antibody .

What are the optimal conditions for using SPBC713.05 antibody in Western blot applications?

Optimal conditions for SPBC713.05 antibody in Western blotting include:

• Sample preparation: S. pombe cells should be lysed in a buffer containing appropriate protease inhibitors. For membrane-associated proteins, detergent selection is critical.

• Blocking solution: 5% non-fat dry milk or 5% BSA in TBST is typically effective. For phospho-specific detection, BSA is preferred as milk contains phosphoproteins.

• Antibody dilution: Start with manufacturer's recommended dilution (typically 1:1000) and optimize based on signal-to-noise ratio.

• Incubation conditions: Primary antibody incubation at 4°C overnight often yields cleaner results than shorter incubations at room temperature.

• Detection system: For low abundance proteins, enhanced chemiluminescence (ECL) or fluorescent secondary antibodies may provide better sensitivity .

Optimization should follow a systematic approach, changing one parameter at a time while keeping others constant .

How do I troubleshoot weak or absent signals when using SPBC713.05 antibody?

When experiencing weak or absent signals with SPBC713.05 antibody, consider these troubleshooting steps:

• Verify protein expression: Confirm that SPBC713.05 is expressed in your chosen cell system under your experimental conditions. Reference transcriptomic data if available.

• Optimize protein extraction: Test different lysis buffers and conditions, as some proteins require specialized extraction methods.

• Increase protein concentration: Load more protein (up to 50-100 μg) to enhance detection of low-abundance proteins.

• Reduce antibody dilution: Use a higher concentration of primary antibody.

• Enhance signal amplification: Use a more sensitive detection system (e.g., enhanced ECL substrate).

• Modify transfer conditions: Adjust transfer time or buffer composition for optimal transfer of proteins to the membrane.

• Test different membrane types: PVDF membranes often provide better protein retention than nitrocellulose for certain applications .

How reliable is SPBC713.05 antibody for detecting protein-protein interactions in co-immunoprecipitation studies?

When using SPBC713.05 antibody for co-immunoprecipitation (co-IP) studies, reliability depends on several factors:

• Antibody affinity: Higher-affinity antibodies generally perform better in capturing native protein complexes.

• Epitope accessibility: Ensure the epitope recognized by the antibody is not masked within protein complexes or by post-translational modifications.

• Validation approach: Before co-IP experiments, validate that the antibody can effectively immunoprecipitate SPBC713.05 from cell lysates using Western blot confirmation.

• Cross-linking considerations: Weak or transient interactions may require chemical cross-linking before immunoprecipitation.

• Buffer optimization: Adjust salt concentration and detergent levels to maintain protein complexes while reducing non-specific binding.

A reciprocal co-IP approach (immunoprecipitating with antibodies against suspected interaction partners) should be used to confirm interactions identified with SPBC713.05 antibody .

Can SPBC713.05 antibody be used for quantitative studies of protein expression levels?

For quantitative studies of SPBC713.05 expression:

• Establish a standard curve: Use purified recombinant SPBC713.05 protein to create a calibration curve.

• Verify linear range: Determine the linear range of detection to ensure measurements are made within this range.

• Use appropriate controls: Include internal loading controls and normalization standards.

• Consider multiple antibodies: Use at least two antibodies recognizing different epitopes to confirm quantitative results.

• Complementary methods: Validate quantitative antibody-based measurements with orthogonal methods like mass spectrometry.

For reproducible quantification, the signal-to-noise ratio and dynamic range are critical parameters, requiring careful optimization of antibody concentration .

How do I assess batch-to-batch variability in SPBC713.05 antibody performance?

To assess batch-to-batch variability:

• Standardized testing: Each new antibody lot should be tested on identical samples used to validate previous lots.

• Quantitative metrics: Compare quantitative parameters including:

  • Signal-to-noise ratio

  • EC50 values in dilution series

  • Band intensity at fixed sample concentrations

  • Background levels

• Reference samples: Maintain a reference sample set that can be used to benchmark new antibody lots.

• Documentation: Keep detailed records of antibody performance using standardized protocols and reference sample sets.

A systematic approach comparing at least 20-40 test samples across different lots is recommended to establish reproducibility metrics .

What standards should I follow when reporting SPBC713.05 antibody use in publications?

When reporting SPBC713.05 antibody use in publications, adhere to these comprehensive standards :

• Antibody identification information:

  • Vendor/source and catalog number

  • Clone name for monoclonal antibodies

  • Host species and antibody type (monoclonal/polyclonal)

  • Research Resource Identifier (RRID) number when available

• Validation methods employed:

  • Detailed description of controls used

  • Reference to validation data (either published or in supplementary materials)

  • Any limitation of validation methods

• Experimental conditions:

  • Complete protocol details including blocking agents, antibody dilutions, incubation times and temperatures

  • Detection method specifications

  • Image acquisition parameters

• Raw data availability:

  • Provide full, uncropped blots in supplementary materials

  • Include positive and negative controls in displayed figures

Publishing these details enhances reproducibility and allows other researchers to properly evaluate and build upon your findings .

How does SPBC713.05 antibody performance compare between different S. pombe strains?

When using SPBC713.05 antibody across different S. pombe strains:

• Strain variability: Expression levels of SPBC713.05 may vary between wild-type strains (e.g., 972 h- vs. h90 strains). Consider strain-specific validation.

• Genetic background effects: Mutations in other genes may affect SPBC713.05 expression or detection. Include appropriate controls for each genetic background.

• Growth conditions: Culture conditions (media, temperature, growth phase) can substantially affect protein expression and detection sensitivity.

• Cross-reactivity assessment: Test antibody specificity across multiple strains, especially when working with engineered strains containing tagged proteins or gene deletions.

Comparative studies should include standardized preparation methods and normalized protein loading to accurately assess antibody performance across strains .

What methodological considerations are important when using SPBC713.05 antibody in studies of cell cycle regulation?

For cell cycle regulation studies with SPBC713.05 antibody:

• Synchronization protocols: Different synchronization methods may affect protein expression, localization, or post-translational modifications. Validate antibody performance under your specific synchronization method.

• Fixation effects: Cell cycle analysis often requires specific fixation methods that may affect epitope recognition. Test multiple fixation protocols to determine optimal conditions.

• Co-localization studies: When examining SPBC713.05 localization throughout the cell cycle, co-staining with known cell cycle markers provides important reference points.

• Temporal resolution: For detailed cell cycle studies, establish a time-course sampling strategy with sufficient temporal resolution to capture changes in protein expression or localization.

• Quantification approaches: Develop standardized methods for quantifying signal intensity throughout the cell cycle to enable statistical analysis of expression patterns .

How does the performance of SPBC713.05 antibody compare to antibodies against related WD repeat proteins?

When comparing SPBC713.05 antibody performance to antibodies against related WD repeat proteins:

• Cross-reactivity assessment: Test for cross-reactivity with other WD repeat proteins, particularly those with high sequence homology to SPBC713.05.

• Epitope mapping: Determine if the epitopes recognized by the antibodies are within conserved WD repeat regions or unique sequences.

• Comparative validation: Apply identical validation methods to all antibodies being compared, including knockout controls and orthogonal detection methods.

• Application-specific comparison: An antibody that performs well in Western blotting may not be optimal for immunofluorescence or chromatin immunoprecipitation.

• Sensitivity and specificity metrics: Compare quantitative metrics like signal-to-noise ratio and detection limits across antibodies under standardized conditions.

This comparative approach can provide insights into both antibody performance and protein function within the WD repeat protein family .

What technical insights can be gained from comparing monoclonal versus polyclonal antibodies against SPBC713.05?

Comparing monoclonal and polyclonal antibodies against SPBC713.05 reveals several important technical considerations:

Understanding these differences allows researchers to select the appropriate antibody type based on experimental requirements and to implement suitable validation strategies for each antibody type .

What emerging technologies might improve SPBC713.05 antibody development and validation?

Several emerging technologies could substantially improve SPBC713.05 antibody development and validation:

• CRISPR-based validation: Using CRISPR-Cas9 to generate knockout cell lines specifically for antibody validation provides gold-standard negative controls .

• Synthetic antibody libraries: Phage or yeast display of synthetic antibody libraries allows selection of high-affinity binders with predetermined specificity profiles.

• Single B-cell sequencing: Isolation and sequencing of single B-cells enables discovery of novel antibody sequences with potentially superior binding characteristics.

• Computational epitope prediction: Advanced algorithms can predict optimal epitopes for antibody development, potentially improving specificity.

• Automated validation platforms: High-throughput systems that test antibody performance across multiple applications and conditions simultaneously.

• Nanobody technology: Development of smaller single-domain antibodies may offer advantages for certain applications, particularly where epitope accessibility is limited.

Implementation of these technologies could address current limitations in antibody reproducibility and specificity .

How might advances in protein structure prediction impact SPBC713.05 antibody design and applications?

Recent advances in protein structure prediction have significant implications for SPBC713.05 antibody research:

• Epitope accessibility analysis: AI-powered structure prediction tools like AlphaFold2 can identify optimally exposed epitopes for antibody development.

• Conformational epitope targeting: Improved structural understanding enables design of antibodies targeting specific protein conformations.

• Cross-reactivity prediction: Structural comparison with related proteins can help predict and minimize potential cross-reactivity.

• Application-specific design: Structure-guided engineering of antibodies optimized for specific applications (e.g., recognizing native vs. denatured forms).

• Functional domain targeting: Antibodies can be designed to target functional domains, potentially enabling modulation of protein activity.

• PTM-specific detection: Structural insights can guide development of antibodies specific to post-translationally modified forms of SPBC713.05.