SPCC965.11c Antibody

What is SPCC965.11c protein and why is it important in fission yeast research?

SPCC965.11c is a protein found in Schizosaccharomyces pombe (strain 972 / ATCC 24843), commonly known as fission yeast. The protein is cataloged in the UniProt database with accession number O59831 . While the specific biological function of SPCC965.11c remains under investigation, studying this protein contributes to our understanding of basic cellular processes in S. pombe, which serves as an important model organism for eukaryotic cell biology research.

The importance of SPCC965.11c antibodies lies in their ability to specifically detect and isolate this protein from complex biological samples, enabling researchers to investigate its expression patterns, cellular localization, interactions with other proteins, and potential roles in cellular processes.

What experimental applications has the SPCC965.11c antibody been validated for?

Based on current literature, SPCC965.11c antibody has been tested and validated for the following applications:

When using this antibody for applications where it hasn't been explicitly validated, researchers should conduct preliminary validation studies to ensure specificity and sensitivity in their particular experimental setup .

What are the optimal storage and handling conditions for SPCC965.11c antibody?

For maximum stability and activity retention of SPCC965.11c antibody:

• Upon receipt, store at -20°C or -80°C to maintain antibody stability

• Avoid repeated freeze-thaw cycles as this can lead to denaturation and loss of activity

• The antibody is supplied in liquid form with 50% glycerol and 0.01M PBS (pH 7.4) containing 0.03% Proclin 300 as a preservative

• For working solutions, aliquot the antibody into smaller volumes before freezing to minimize freeze-thaw cycles

• When handling, use sterile techniques and avoid contamination

How can I validate the specificity of SPCC965.11c antibody in my experimental system?

Validating antibody specificity is crucial for generating reliable research data. For SPCC965.11c antibody, consider implementing this validation workflow:

• Genetic validation approach (preferred method):

  • Test the antibody on samples from both wild-type and SPCC965.11c knockout S. pombe strains

  • A specific antibody should show signal in wild-type samples but not in knockout samples

  • This genetic approach has been shown to be more reliable than orthogonal methods, particularly for immunofluorescence applications

• Immunoblot analysis:

  • Run protein lysates from S. pombe alongside a negative control (non-yeast sample)

  • A specific single band at the expected molecular weight indicates specificity

  • Multiple bands may indicate cross-reactivity with other proteins

• Peptide competition assay:

  • Pre-incubate the antibody with excess recombinant SPCC965.11c protein

  • This should abolish specific binding in your experimental samples

  • This method can help confirm that the observed signal is due to specific binding

• Mass spectrometry validation:

  • Perform immunocapture with the antibody followed by mass spectrometry

  • Analysis should identify peptides from SPCC965.11c as the predominant hits

  • This is considered a highly reliable validation method for immunocapture applications

Recent studies on antibody validation have shown that approximately 50% of commercial antibodies fail in one or more validation tests, highlighting the importance of thorough validation before use in critical experiments .

What controls should I include when using SPCC965.11c antibody in my experiments?

Incorporating appropriate controls is essential for interpreting results obtained with SPCC965.11c antibody:

Remember that genetic knockout controls have been demonstrated to be more reliable than orthogonal approaches for antibody validation . In cases where knockout controls are unavailable, using multiple distinct antibodies targeting different epitopes of SPCC965.11c can increase confidence in your results .

How do I determine the optimal concentration of SPCC965.11c antibody for Western blotting and immunostaining?

Determining the optimal antibody concentration is crucial for achieving the best signal-to-noise ratio. For SPCC965.11c antibody:

• Perform a titration experiment:

  • Test a range of antibody dilutions (e.g., 1:100, 1:500, 1:1000, 1:5000)

  • Use consistent amounts of protein lysate or identical sample preparation

  • Evaluate based on signal-to-noise ratio rather than absolute signal intensity

• For Western blotting:

  • Start with manufacturer's recommended dilution (if provided)

  • Optimal concentration should give clear specific bands with minimal background

  • Too high concentration can yield non-specific binding; too low can lead to false-negative results

• For immunostaining:

  • Begin with 1-5 μg/mL range if no recommendation is available

  • Pay particular attention to antigen retrieval methods for fixed samples

  • Test different retrieval methods if initial results are unsatisfactory

  • Note that optimal antibody concentration may need adjustment when changing retrieval methods

• Quantitative analysis:

  • Calculate signal-to-noise ratio by dividing specific signal intensity by background

  • Plot signal-to-noise ratio against antibody concentration to identify optimal dilution

  • At the optimal concentration, doubling the amount should still maintain specificity

Can SPCC965.11c antibody be used effectively in immunoprecipitation experiments?

While the SPCC965.11c antibody has not been explicitly validated for immunoprecipitation according to the available data , many polyclonal antibodies can perform well in IP experiments due to their recognition of multiple epitopes. When adapting this antibody for immunoprecipitation:

• Optimization strategy:

  • Test different binding conditions (varying buffer compositions, pH, salt concentrations)

  • Compare IP efficiency with different antibody amounts (typically 1-5 μg per reaction)

  • Consider crosslinking the antibody to beads to prevent antibody contamination in eluates

• Validation approach:

  • Perform IP followed by Western blot with the same or a different validated SPCC965.11c antibody

  • For conclusive validation, analyze immunoprecipitated proteins by mass spectrometry

  • A successful IP should show enrichment of SPCC965.11c peptides compared to control IPs

• Technical considerations:

  • The antibody is affinity-purified, which generally increases specificity in IP applications

  • Being a polyclonal IgG antibody may provide advantage in capturing native protein complexes

  • Non-conjugated format allows flexibility in choosing immunoprecipitation protocol

Recent large-scale antibody validation studies have shown that approximately 43.6% of antibodies pass quality control for immunoprecipitation applications, with recombinant antibodies generally outperforming polyclonal antibodies in this application .

How can computational approaches aid in optimizing SPCC965.11c antibody-based experiments?

Modern computational tools can enhance the design and interpretation of antibody-based experiments:

• Epitope prediction and analysis:

  • Use structural bioinformatics tools to predict the epitope recognized by the antibody

  • Analyze potential cross-reactivity with related proteins through sequence alignment

  • Programs like RosettaAntibodyDesign (RAbD) can model antibody-antigen interactions

• Structure-based optimization:

  • If the three-dimensional structure of SPCC965.11c is available or can be modeled:

    • Predict antibody-antigen complex structures

    • Identify potential steric hindrances in certain applications

    • Optimize binding conditions based on structural insights

• Data mining from antibody databases:

  • Utilize databases like PLAbDab (Patent and Literature Antibody Database)

  • Compare with similar antibodies against related proteins for protocol optimization

  • These resources contain over 150,000 paired antibody sequences that can provide valuable insights

• Benchmark against validated antibodies:

  • Compare performance metrics with well-characterized antibodies

  • Standardize validation protocols based on computational benchmarking

  • This approach has been shown to improve antibody selection process

Computational approaches have demonstrated value in antibody engineering, with in silico methods successfully improving antibody affinities and physicochemical properties in multiple studies .

What are the considerations for multiplexing SPCC965.11c antibody with other antibodies in co-localization studies?

Multiplexing antibodies requires careful planning to avoid technical artifacts:

• Antibody compatibility assessment:

  • Ensure primary antibodies are raised in different host species to avoid cross-reactivity

  • Test for potential cross-reactivity between secondary antibodies

  • Validate that fixation and permeabilization methods are compatible with all antibodies in the panel

• Sequential immunostaining strategy:

  • For challenging combinations, consider sequential rather than simultaneous staining

  • Block between rounds using excess unconjugated secondary antibody

  • Validate that the first round of staining isn't affected by subsequent rounds

• Spectral considerations:

  • Choose fluorophores with minimal spectral overlap

  • Include single-stain controls for spectral unmixing if needed

  • If using directly conjugated antibodies, verify that conjugation hasn't affected binding properties

• Validation of co-localization:

  • Use quantitative co-localization metrics (Pearson's correlation coefficient, Manders' overlap coefficient)

  • Include control samples where co-localization is expected and where it is not

  • Perform z-stack imaging to verify true co-localization in three dimensions

What are common causes of high background when using SPCC965.11c antibody, and how can they be addressed?

High background can significantly reduce data quality in antibody-based experiments. For SPCC965.11c antibody:

Recent studies on antibody validation suggest that approximately 63.5% of antibodies fail quality control for immunofluorescence applications, highlighting the importance of optimization in this technique .

How can I improve reproducibility when using SPCC965.11c antibody across different experimental batches?

Achieving consistent results with antibodies requires systematic approach:

• Standardize sample preparation:

  • Use consistent cell growth conditions and lysis procedures

  • Standardize protein quantification methods

  • Process all comparative samples simultaneously when possible

• Maintain antibody quality:

  • Aliquot antibody upon receipt to minimize freeze-thaw cycles

  • Document lot numbers and compare performance between lots

  • Consider dedicated antibody validation for each new lot

• Quantitative analysis:

  • Use digital image analysis rather than visual assessment

  • Include standard curves where appropriate

  • Normalize to reliable loading controls or internal standards

• Experimental design considerations:

  • Include biological and technical replicates

  • Randomize sample order during processing

  • Use blinded analysis where possible to reduce bias

• Documentation practices:

  • Maintain detailed protocols including all buffer compositions

  • Record all experimental parameters including incubation times and temperatures

  • Document any deviations from standard protocols

Recent antibody validation initiatives have highlighted that standardized protocols and comprehensive documentation are critical for addressing the "reproducibility crisis" in antibody-based research .

How should I approach contradictory results between SPCC965.11c antibody detection and other experimental methods?

When faced with discrepancies between antibody-based results and other techniques:

• Systematic validation:

  • Re-validate antibody specificity using genetic approaches (knockout controls)

  • Test multiple antibodies targeting different epitopes of SPCC965.11c

  • Consider that genetic validation approaches have been shown to be more reliable than orthogonal approaches

• Technical considerations:

  • Different techniques detect proteins in different states (denatured vs. native)

  • Post-translational modifications may affect antibody recognition

  • Protein complex formation may mask epitopes in certain assays

• Complementary approach:

  • Use orthogonal methods to corroborate findings (e.g., mass spectrometry)

  • Consider RNA-level analysis (qPCR, RNA-seq) alongside protein detection

  • Employ functional assays when appropriate

• Critical analysis:

  • Evaluate the sensitivity and specificity of each method

  • Consider sample preparation differences between techniques

  • Assess the limitations and assumptions of each method

Recent large-scale analyses of antibodies have shown that approximately 20-30% of figures in published literature may be generated using antibodies that do not recognize their intended targets, highlighting the importance of thorough validation when discrepancies arise .

How might high-throughput antibody validation methods improve our confidence in SPCC965.11c antibody results?

Emerging high-throughput validation approaches offer promising avenues for enhanced antibody reliability:

• CRISPR-Cas9 knockout validation panels:

  • Development of systematic knockout cell lines for antibody validation

  • Standardized testing across multiple applications (WB, IP, IF)

  • This approach has proven valuable in recent large-scale validation studies

• Automated imaging and analysis pipelines:

  • Standardized immunofluorescence protocols with quantitative readouts

  • Machine learning algorithms for specificity assessment

  • Reduced human bias in interpretation of results

• Proteome-wide specificity assessment:

  • Mass spectrometry identification of all proteins captured by an antibody

  • Quantitative ranking of specific vs. non-specific binding

  • Integration with antibody databases for cross-comparison

• Community-based validation resources:

  • Centralized databases documenting antibody performance

  • Standard validation protocols adopted across research communities

  • Open sharing of validation data through repositories like ZENODO

Initiatives like YCharOS have evaluated approximately 1000 antibodies against around 100 targets, providing open head-to-head comparisons using standardized protocols, and have led to manufacturers removing or modifying recommendations for over 200 underperforming antibodies .

What emerging applications might utilize SPCC965.11c antibody beyond traditional techniques?

As antibody technologies continue to evolve, several cutting-edge applications may be possible:

• Single-cell analysis techniques:

  • Mass cytometry (CyTOF) for high-dimensional protein profiling

  • Imaging mass cytometry for spatial protein localization

  • Integration with single-cell RNA sequencing for multi-omic analysis

• Advanced microscopy applications:

  • Super-resolution microscopy for nanoscale localization

  • Live-cell imaging with nanobody derivatives

  • Expansion microscopy for enhanced spatial resolution

• Proximity-based interaction studies:

  • Proximity ligation assays for protein-protein interaction detection

  • BioID or APEX2 proximity labeling when fused to nanobodies

  • FRET-based interaction studies for dynamic protein complexes

• Therapeutic and diagnostic development:

  • If SPCC965.11c has human homologs, antibody engineering approaches like RosettaAntibodyDesign could be applied

  • Sequential design-build-test cycles to generate optimized antibodies with enhanced properties

The increasing integration of computational methods with experimental validation, as seen in recent antibody engineering studies, provides a framework for developing antibodies with improved specificity and sensitivity .