SPBC146.08c Antibody

What is SPBC146.08c protein and why is it studied?

SPBC146.08c is a protein encoded in Schizosaccharomyces pombe (strain 972/ATCC 24843), commonly known as fission yeast. This protein has gained research interest due to its potential role in fundamental cellular processes. While specific function details are still being investigated, researchers utilize this antibody to explore protein expression, localization, and interactions within the S. pombe model organism. Fission yeast serves as an excellent eukaryotic model due to its genetic tractability and conservation of many basic cellular processes found in higher eukaryotes, including humans .

What are the basic specifications of commercially available SPBC146.08c antibodies?

The SPBC146.08c antibody is typically available as a polyclonal antibody raised in rabbits. Current specifications include:

The antibody is specifically designed for research applications and should not be used for diagnostic or therapeutic purposes .

What are the recommended primary applications for SPBC146.08c antibody?

The SPBC146.08c antibody has been validated for several research applications:

• Western Blotting (WB): For detecting SPBC146.08c protein expression levels and molecular weight verification in cell lysates and tissue homogenates. This application allows for semi-quantitative analysis of protein expression.

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of SPBC146.08c protein in samples. This application is particularly useful for high-throughput screening.

Both applications have been validated to ensure proper identification of the antigen of interest . Similar to other research antibodies like those in the DSHB collection, researchers should follow standard protocols for these applications while optimizing conditions for this specific antibody .

How should researchers optimize Western blot protocols for SPBC146.08c detection?

Optimizing Western blot protocols for SPBC146.08c requires careful consideration of several parameters:

• Sample Preparation:

  • For S. pombe cells, use glass bead or enzymatic lysis methods with protease inhibitors

  • Typical loading concentration: 20-50 μg total protein per lane

  • Include positive and negative controls

• Gel Selection and Separation:

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

  • Consider gradient gels for better resolution

• Transfer Optimization:

  • Semi-dry or wet transfer at 100V for 60-90 minutes

  • Use PVDF membranes (0.45 μm) for better protein retention

• Blocking and Antibody Dilution:

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

  • Test antibody dilutions ranging from 1:500 to 1:2000

  • Incubate primary antibody overnight at 4°C for best results

• Detection Method:

  • Use HRP-conjugated secondary antibodies with enhanced chemiluminescence

  • Consider signal enhancement systems for low abundance proteins

Similar optimization approaches have been documented for other yeast protein antibodies, and these principles can be applied to SPBC146.08c detection with appropriate modifications .

What cross-reactivity concerns should researchers consider when using SPBC146.08c antibody?

Cross-reactivity is a critical consideration for antibody specificity and experimental validity:

• Species Cross-reactivity:

  • The antibody is specifically developed against S. pombe SPBC146.08c

  • Cross-reactivity with orthologs in related yeast species (S. cerevisiae, C. albicans) is possible but requires validation

  • Human or other mammalian cross-reactivity is unlikely but should be assessed if using in comparative studies

• Validation Methods for Cross-reactivity:

  • Knockout/knockdown controls: Use S. pombe strains with SPBC146.08c deletions

  • Peptide competition assays: Pre-incubate antibody with recombinant SPBC146.08c

  • Multiple antibody approach: Compare results with other antibodies targeting different epitopes

• Nonspecific Binding Mitigation:

  • Increase washing steps and duration

  • Optimize blocking conditions

  • Use highly purified antibody preparations

This methodical approach to cross-reactivity assessment follows established antibody validation practices, similar to those used for other research antibodies like Sp14 and Sp-40C .

How can researchers effectively use SPBC146.08c antibody for immunofluorescence studies?

While not explicitly listed among validated applications, many polyclonal antibodies can be adapted for immunofluorescence studies following these optimization steps:

• Fixation Protocol Development:

  • Test multiple fixation methods: 4% paraformaldehyde, methanol/acetone, or specialized yeast wall digestion protocols

  • Optimize fixation time (10-30 minutes) and temperature

• Permeabilization Strategies:

  • S. pombe cell wall requires specialized permeabilization

  • Test enzymatic digestion (zymolyase, lysing enzymes) followed by 0.1-0.5% Triton X-100

  • Optimize concentrations and incubation times

• Antibody Concentration Determination:

  • Start with higher concentrations (1:50-1:200) for initial tests

  • Titrate to optimize signal-to-noise ratio

  • Include appropriate controls (secondary-only, pre-immune serum)

• Signal Enhancement:

  • Consider tyramide signal amplification for low-abundance proteins

  • Use high-sensitivity detection systems

• Counterstaining:

  • DAPI for nuclear visualization

  • Cell wall stains (calcofluor white) for morphological context

This approach incorporates best practices from successful immunofluorescence studies with other yeast proteins and antibodies, such as the Sp14 antibody which is specifically recommended for immunofluorescence applications .

How should researchers design experiments to quantify SPBC146.08c expression levels across different conditions?

Experimental design for quantitative analysis of SPBC146.08c expression requires careful planning:

• Experimental Design Framework:

  • Include biological replicates (minimum n=3) for statistical validity

  • Incorporate technical replicates within each biological sample

  • Design appropriate time course experiments for dynamic expression analysis

• Controls and Normalization Strategy:

  • Use constitutively expressed proteins (tubulin, actin) as loading controls

  • Consider spike-in controls for absolute quantification

  • Include positive controls from conditions known to express SPBC146.08c

• Data Collection Methods:

  • For Western blot: Use digital image capture with linear dynamic range

  • For ELISA: Generate standard curves with recombinant protein

  • Consider qPCR for mRNA levels alongside protein detection

• Statistical Analysis Approach:

  • Apply appropriate statistical tests (t-test, ANOVA) based on experimental design

  • Use non-parametric tests if assumptions of normality cannot be met

  • Calculate confidence intervals to report variability

• Data Visualization:

  • Present normalized expression levels with error bars

  • Use consistent scaling for comparable conditions

  • Consider heat maps for complex experimental designs

This structured approach ensures reproducibility and statistical rigor in expression analysis, similar to established methods used for other research antibodies in model organisms .

What are the recommended methods for validating the specificity of SPBC146.08c antibody in research applications?

Validating antibody specificity is crucial for research integrity and reproducibility:

• Genetic Validation:

  • Compare staining/detection between wild-type and SPBC146.08c deletion strains

  • Use CRISPR/Cas9 knockout systems if traditional deletion strains are unavailable

  • Test inducible expression systems to observe corresponding signal changes

• Biochemical Validation:

  • Perform peptide competition assays with the immunizing antigen

  • Conduct immunoprecipitation followed by mass spectrometry

  • Compare detection patterns using different antibodies targeting the same protein

• Specificity Controls:

  • Pre-immune serum controls to assess background

  • Isotype controls to distinguish specific from non-specific binding

  • Secondary antibody-only controls to evaluate background

• Cross-platform Validation:

  • Correlate Western blot results with ELISA quantification

  • Compare protein detection with mRNA expression (qPCR, RNA-seq)

  • Verify localization with tagged protein constructs

These validation approaches follow best practices established for research antibodies and are essential for ensuring experimental reproducibility .

What are common issues encountered with SPBC146.08c antibody in Western blotting and how can they be resolved?

Researchers may encounter several challenges when using SPBC146.08c antibody in Western blotting:

• Weak or No Signal:

  • Problem: Insufficient antibody concentration or protein expression

  • Solution: Increase antibody concentration; optimize protein extraction; use enhanced chemiluminescence detection; increase exposure time

  • Methodological Approach: Employ step-gradient antibody dilutions (1:500, 1:1000, 1:2000) to determine optimal concentration

• High Background:

  • Problem: Non-specific binding or insufficient blocking/washing

  • Solution: Increase blocking time/concentration; use alternative blocking agents (BSA vs. milk); increase wash duration/frequency; dilute antibody in fresh blocking solution

  • Methodological Approach: Systematically test different blocking agents and washing protocols

• Multiple Bands:

  • Problem: Cross-reactivity, protein degradation, or post-translational modifications

  • Solution: Use freshly prepared samples with protease inhibitors; optimize lysis conditions; verify with knockout controls

  • Methodological Approach: Compare band patterns between wild-type and mutant strains

• Inconsistent Results:

  • Problem: Variability in sample preparation or antibody performance

  • Solution: Standardize protocols; aliquot antibody to avoid freeze-thaw cycles; include positive controls in each experiment

  • Methodological Approach: Develop detailed SOPs for all experimental steps

These troubleshooting approaches are based on general antibody optimization principles and can be applied specifically to SPBC146.08c antibody work .

How can researchers optimize storage and handling of SPBC146.08c antibody to maintain long-term activity?

Proper storage and handling are critical for maintaining antibody performance:

• Initial Processing:

  • Upon receipt, prepare small working aliquots (20 μl minimum) to minimize freeze-thaw cycles

  • For short-term use (within two weeks), store at 4°C

  • For long-term storage, keep at -20°C or preferably -80°C

• Storage Preparation:

  • Consider adding equal volume of glycerol as cryoprotectant before freezing

  • Use sterile, low-protein binding tubes for storage

  • Label tubes with antibody details, concentration, and date

• Handling Best Practices:

  • Allow aliquots to warm to room temperature before opening to prevent condensation

  • Centrifuge briefly before opening tubes

  • Use clean pipette tips to prevent contamination

  • Return to storage promptly after use

• Activity Monitoring:

  • Include positive controls in each experiment to monitor antibody performance

  • Compare signal intensity over time to detect potential activity loss

  • Document lot numbers and correlate with experimental outcomes

These recommendations align with established storage practices for research antibodies as documented in DSHB resources and can be applied to maintain SPBC146.08c antibody quality .

How can SPBC146.08c antibody be used in chromatin immunoprecipitation (ChIP) studies?

While not explicitly validated for ChIP, researchers can adapt the SPBC146.08c antibody for this application:

• Protocol Adaptation Requirements:

  • Cross-linking optimization: Test different formaldehyde concentrations (0.75-1.5%) and times (5-20 minutes)

  • Chromatin fragmentation: Standardize sonication conditions to achieve 200-500 bp fragments

  • IP conditions: Higher antibody concentrations (5-10 μg per reaction) may be required

  • Washing stringency: Develop specific washing protocols to minimize background

• Controls Design:

  • Input controls: Essential for normalization

  • IgG controls: Match host species (rabbit) for background assessment

  • Positive controls: Include antibodies against histones or known DNA-binding proteins

  • Negative controls: Target regions not expected to associate with SPBC146.08c

• Validation Approach:

  • qPCR validation of enriched regions before proceeding to sequencing

  • Assessment of signal-to-noise ratio across multiple experimental conditions

  • Comparison with tagged protein approaches (if available)

• Data Analysis Considerations:

  • Use specialized ChIP-seq analysis pipelines

  • Apply appropriate normalization methods

  • Consider replicate concordance analysis

This methodological framework draws on established ChIP protocols from research with other antibodies, adapted specifically for yeast chromatin studies .

What considerations are important when using SPBC146.08c antibody for co-immunoprecipitation (Co-IP) studies?

Co-immunoprecipitation requires specific optimization for successful protein-protein interaction studies:

• Lysis Buffer Optimization:

  • Test multiple lysis conditions: from gentle (low salt, mild detergents) to stringent

  • Consider specialized yeast lysis protocols with enzymatic pre-treatment

  • Include protease and phosphatase inhibitors to maintain protein interactions

  • Evaluate different detergents (NP-40, Triton X-100, CHAPS) at varying concentrations

• Antibody Coupling Strategies:

  • Direct approach: Antibody added to lysate, followed by Protein A/G beads

  • Pre-coupling approach: Antibody bound to beads before lysate addition

  • Crosslinking consideration: Evaluate whether crosslinking antibody to beads improves results

• Experimental Controls:

  • Input samples (5-10% of starting material)

  • IgG control immunoprecipitations

  • Reciprocal Co-IPs when possible

  • Knockout/knockdown controls

• Interaction Validation Methods:

  • Repeat experiments under different conditions

  • Confirm interactions using alternative methods (proximity ligation, Y2H)

  • Consider size exclusion chromatography as complementary approach

Similar methodological approaches have been successful with other research antibodies for immunoprecipitation studies, such as the PCRP-ASCL4-2G8 antibody which is specifically recommended for such applications .

How can researchers integrate SPBC146.08c antibody data with other -omics approaches?

Multi-omics integration provides comprehensive understanding of biological systems:

• Proteomics Integration:

  • Compare antibody-based quantification with mass spectrometry data

  • Correlate post-translational modifications detected by specific antibodies with proteomics datasets

  • Use antibody-based enrichment prior to MS analysis for targeted proteomics

• Transcriptomics Correlation:

  • Compare protein levels (Western blot, ELISA) with mRNA expression (RNA-seq, qPCR)

  • Investigate potential post-transcriptional regulation mechanisms when discrepancies occur

  • Develop integrated models of gene expression regulation

• Functional Genomics Connections:

  • Correlate protein localization/expression with phenotypic data from genetic screens

  • Integrate ChIP-seq data with transcriptome analysis for regulatory network reconstruction

  • Map protein interactions to functional pathways

• Data Integration Tools and Approaches:

  • Use specialized software for multi-omics data integration

  • Apply machine learning algorithms to identify patterns across datasets

  • Develop visualization strategies for complex data relationships

This integrated approach enables researchers to place SPBC146.08c studies in broader biological context, similar to integrative approaches used with other research antibodies .

What are the current limitations of SPBC146.08c antibody research and how might these be addressed?

Current research with SPBC146.08c antibody faces several limitations:

• Application Range Limitations:

  • Currently validated only for ELISA and Western blot applications

  • Limited data on immunohistochemistry, flow cytometry, and other advanced applications

  • Future Direction: Systematic validation across multiple applications using standardized protocols

• Cross-reactivity Documentation:

  • Incomplete characterization of potential cross-reactivity with related proteins

  • Limited testing across diverse experimental conditions

  • Future Direction: Comprehensive cross-reactivity testing with closely related proteins and across species

• Reproducibility Challenges:

  • Batch-to-batch variation inherent to polyclonal antibodies

  • Limited standardization of validation protocols

  • Future Direction: Development of monoclonal alternatives or recombinant antibodies

• Functional Characterization Gaps:

  • Incomplete understanding of SPBC146.08c protein function

  • Limited integrative studies connecting antibody-based detection with functional outcomes

  • Future Direction: Systematic functional studies correlating protein detection with phenotypic outcomes

These limitations and proposed solutions align with broader challenges in research antibody development and application, as seen with other research antibodies in the field .

How might emerging antibody technologies enhance SPBC146.08c research in the future?

Emerging technologies offer new possibilities for SPBC146.08c research:

• Next-Generation Antibody Formats:

  • Recombinant antibodies with consistent production and reduced batch variation

  • Single-domain antibodies (nanobodies) for improved access to epitopes

  • Synthetic affinity reagents with customizable binding properties

• Advanced Detection Systems:

  • Super-resolution microscopy compatible antibody conjugates

  • Multiplexed detection systems for simultaneous visualization of multiple targets

  • Quantitative single-molecule detection approaches

• Functional Antibody Applications:

  • Intrabodies for live-cell tracking of SPBC146.08c

  • Proximity-dependent labeling for interaction mapping

  • Optogenetic antibody systems for spatiotemporal control

• AI-Enhanced Antibody Development:

  • Computational epitope prediction for improved antibody design

  • Machine learning approaches to optimize antibody performance

  • In silico screening to reduce cross-reactivity

These technological advances represent the future direction of antibody research tools, building upon the foundation of current antibody technologies while addressing their limitations .