SPBC19G7.04 Antibody

What is SPBC19G7.04 and why is it significant in S. pombe research?

SPBC19G7.04 is a protein found in Schizosaccharomyces pombe (fission yeast) with the UniProt accession number O42953. The antibody against this protein is significant in S. pombe research because it enables specific detection of this protein in various experimental contexts. S. pombe serves as an important model organism in molecular and cellular biology research, particularly for studying cell cycle regulation, DNA damage responses, and other conserved cellular processes. The SPBC19G7.04 antibody allows researchers to track the expression, localization, and behavior of this specific protein within the experimental system .

What are the optimal storage conditions for SPBC19G7.04 antibody to maintain reactivity?

For optimal preservation of SPBC19G7.04 antibody reactivity, store the antibody at -20°C or -80°C upon receipt. The antibody is supplied in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. This formulation helps maintain stability during storage. Avoid repeated freeze-thaw cycles, as these can lead to protein denaturation and loss of antibody function. When working with the antibody, keep it on ice and return to storage promptly after use. For long-term projects requiring multiple uses, consider aliquoting the antibody into smaller volumes to minimize freeze-thaw cycles .

What applications has the SPBC19G7.04 antibody been validated for?

The SPBC19G7.04 antibody has been validated for specific applications including ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) for identification of the antigen. These applications allow researchers to detect and quantify the SPBC19G7.04 protein in various experimental settings. The antibody's specificity for Schizosaccharomyces pombe (strain 972 / ATCC 24843) makes it particularly valuable for focused studies in this model organism. When designing experiments, researchers should optimize dilutions for each specific application, as optimal concentrations may vary depending on the experimental setup, sample preparation methods, and detection systems used .

How can I ensure proper validation of SPBC19G7.04 antibody specificity in my experimental system?

Validating antibody specificity for SPBC19G7.04 requires a multi-faceted approach:

• Positive and negative controls: Use wild-type S. pombe strains as positive controls and SPBC19G7.04 deletion mutants as negative controls in Western blots and immunostaining.

• Competitive binding assays: Pre-incubate the antibody with purified recombinant SPBC19G7.04 protein before application to verify that binding is blocked, confirming specificity.

• Orthogonal validation: Correlate antibody-based detection with other methods such as mass spectrometry or RNA expression data.

• Cross-reactivity testing: Test the antibody against closely related proteins to ensure it doesn't cross-react with other S. pombe proteins.

• Epitope mapping: If possible, determine the specific epitope recognized by the antibody to understand potential limitations in detecting modified or truncated forms of the protein.

For polyclonal antibodies like the SPBC19G7.04 antibody, batch-to-batch variation should be considered, and new lots should be re-validated before use in critical experiments .

What are the considerations for optimizing SPBC19G7.04 antibody use in immunoprecipitation studies?

While immunoprecipitation (IP) is not listed among the validated applications for the SPBC19G7.04 antibody, researchers interested in adapting it for IP should consider the following optimization strategies:

• Buffer composition optimization: Test various lysis and binding buffers with different salt concentrations (150-500 mM NaCl), detergents (0.1-1% Triton X-100, NP-40, or CHAPS), and pH values (6.8-8.0) to identify conditions that preserve the antigen-antibody interaction while efficiently lysing cells.

• Antibody concentration titration: Determine the optimal antibody amount (typically 1-10 μg per sample) through titration experiments.

• Cross-linking considerations: For polyclonal antibodies like SPBC19G7.04, consider cross-linking the antibody to protein A/G beads using dimethyl pimelimidate (DMP) to prevent antibody leaching during elution.

• Pre-clearing samples: Pre-clear lysates with protein A/G beads without antibody to reduce non-specific binding.

• Elution conditions: Test different elution methods, including low pH (glycine buffer, pH 2.5-3.0), high pH, competitive elution with the immunizing peptide, or direct boiling in SDS sample buffer.

• Validation of results: Confirm IP results using reciprocal IP with antibodies against known interaction partners or through mass spectrometry analysis of co-immunoprecipitated proteins.

Each of these parameters should be systematically tested and optimized for the specific experimental conditions and cell lysis methods used with S. pombe .

How do polyclonal antibodies like SPBC19G7.04 compare with monoclonal antibodies in detecting conformational changes in target proteins?

Polyclonal antibodies like the SPBC19G7.04 antibody offer distinct advantages for detecting conformational changes in target proteins compared to monoclonal antibodies:

The SPBC19G7.04 antibody, being a rabbit polyclonal antibody, can recognize multiple epitopes on the target protein, making it potentially valuable for detecting the protein under various experimental conditions where conformational changes might occur. This characteristic is particularly useful in studies investigating protein dynamics, post-translational modifications, or protein-protein interactions that might alter protein conformation .

What is the recommended protocol for using SPBC19G7.04 antibody in Western blotting of S. pombe lysates?

Western Blotting Protocol for SPBC19G7.04 Detection in S. pombe:

• Sample Preparation:

  • Harvest S. pombe cells in mid-log phase (OD600 ≈ 0.5-0.8)

  • Lyse cells in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail

  • Disrupt cells using glass beads (5 cycles of 1 min vortexing followed by 1 min on ice)

  • Clear lysate by centrifugation at 13,000 × g for 15 minutes at 4°C

• Protein Separation:

  • Determine protein concentration using Bradford or BCA assay

  • Load 20-40 μg of total protein per lane on 10-12% SDS-PAGE gel

  • Include positive control (wild-type S. pombe extract) and negative control (SPBC19G7.04 deletion strain if available)

  • Separate proteins at 120V until adequate resolution is achieved

• Transfer and Blocking:

  • Transfer proteins to PVDF membrane (0.45 μm) at 100V for 1 hour in cold transfer buffer

  • Block membrane with 5% non-fat dry milk in TBST (TBS + 0.1% Tween-20) for 1 hour at room temperature

• Antibody Incubation:

  • Dilute SPBC19G7.04 antibody 1:500 to 1:2000 in 5% BSA in TBST (optimize for each experiment)

  • Incubate membrane with primary antibody overnight at 4°C with gentle rocking

  • Wash 4 times with TBST for 5 minutes each

  • Incubate with HRP-conjugated anti-rabbit IgG secondary antibody (1:5000) for 1 hour at room temperature

  • Wash 4 times with TBST for 5 minutes each

• Detection:

  • Apply ECL substrate and detect signal using film or digital imaging system

  • Expected band size should be verified based on theoretical molecular weight of SPBC19G7.04

• Validation and Controls:

  • Confirm specificity by comparing with expected molecular weight

  • Include loading control (anti-tubulin or anti-GAPDH) on the same blot

  • For challenging detections, consider signal enhancement systems or more sensitive substrates

Always optimize antibody dilution for each new lot to ensure consistent results .

How can I implement dual immunofluorescence staining with SPBC19G7.04 antibody and other S. pombe markers?

While immunofluorescence is not specifically listed among the validated applications for the SPBC19G7.04 antibody, researchers may adapt the following protocol for dual immunofluorescence staining in S. pombe, drawing from general principles of antibody-based immunofluorescence techniques:

Dual Immunofluorescence Protocol:

• Cell Preparation:

  • Grow S. pombe to mid-log phase (OD600 = 0.5-0.8)

  • Fix cells with 3.7% formaldehyde for 30 minutes at room temperature

  • Wash 3 times with PEM buffer (100 mM PIPES pH 6.9, 1 mM EGTA, 1 mM MgSO4)

  • Digest cell walls with zymolyase (1 mg/ml in PEMS + 1.2 M sorbitol) for 30-60 minutes at 37°C

  • Permeabilize with 1% Triton X-100 in PEM for 5 minutes

• Blocking and Primary Antibody Incubation:

  • Block with PEMBAL (PEM + 1% BSA, 0.1% sodium azide, 100 mM lysine hydrochloride) for 30 minutes

  • Dilute SPBC19G7.04 antibody (1:100 to 1:500, optimize for your conditions) and the second marker antibody in PEMBAL

  • Ensure the second antibody is raised in a different species (e.g., mouse) to prevent cross-reactivity

  • Incubate overnight at 4°C in a humid chamber

• Secondary Antibody Incubation:

  • Wash 3 times with PEMBAL for 5 minutes each

  • Apply species-specific secondary antibodies with non-overlapping fluorophores:

    • Anti-rabbit IgG (for SPBC19G7.04) conjugated to Alexa Fluor 488

    • Appropriate secondary for the second primary antibody (e.g., anti-mouse IgG with Alexa Fluor 594)

  • Incubate for 2 hours at room temperature in the dark

  • Wash 3 times with PBS

• Nuclear Staining and Mounting:

  • Counterstain with DAPI (1 μg/ml) for 5 minutes

  • Mount slides with anti-fade mounting medium

  • Seal with nail polish and store at 4°C in the dark

• Controls and Validation:

  • Include single antibody controls to check for bleed-through

  • Use peptide competition controls to confirm specificity

  • Include SPBC19G7.04 deletion strains as negative controls

  • Consider co-localization analysis with known markers for specific subcellular compartments

This protocol should be optimized based on the specific properties of the SPBC19G7.04 antibody and the co-staining marker .

What are the key considerations for using SPBC19G7.04 antibody in ELISA assays?

Since ELISA is one of the validated applications for the SPBC19G7.04 antibody, here is a detailed methodology and optimization guide:

ELISA Protocol and Optimization for SPBC19G7.04 Antibody:

• Assay Format Selection:

  • Direct ELISA: Coat plates with S. pombe lysate or purified SPBC19G7.04 protein

  • Sandwich ELISA: Use a capture antibody against another epitope of SPBC19G7.04 or a tag if using recombinant protein

  • Competitive ELISA: For quantitative analysis or when sample purity is a concern

• Plate Coating Optimization:

  • Protein concentration: Test range from 1-10 μg/ml of total protein from S. pombe lysate

  • Buffer composition: Compare carbonate buffer (pH 9.6) vs. PBS (pH 7.4)

  • Incubation time: Test overnight at 4°C vs. 2 hours at room temperature

• Blocking Optimization:

  • Test different blocking agents: 1-5% BSA, 1-5% non-fat dry milk, or commercial blocking buffers

  • Blocking time: 1-2 hours at room temperature

• Antibody Dilution Optimization:

  • Create a dilution series (1:100 to 1:10,000) of SPBC19G7.04 antibody

  • Generate a standard curve to determine optimal working dilution

  • Determine optimal incubation time (1-2 hours at room temperature or overnight at 4°C)

• Detection System Selection:

  • HRP-conjugated anti-rabbit IgG for colorimetric detection (TMB substrate)

  • AP-conjugated anti-rabbit IgG for enhanced sensitivity (pNPP substrate)

  • Consider streptavidin-biotin amplification systems for lower abundance targets

• Assay Validation Parameters:

  • Specificity: Test against negative controls (SPBC19G7.04 deletion strains)

  • Sensitivity: Determine limit of detection and quantification

  • Linearity: Assess signal proportionality across a range of sample dilutions

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

  • Accuracy: Spike recovery tests with known quantities of recombinant protein

• Data Analysis Considerations:

  • Use appropriate curve-fitting models for standard curves

  • Include calculation of sample concentrations with confidence intervals

  • Consider normalization strategies for comparing across different experiments

What strategies can be employed when SPBC19G7.04 antibody shows weak or inconsistent signals in Western blots?

When encountering weak or inconsistent signals with SPBC19G7.04 antibody in Western blotting, consider these systematic troubleshooting approaches:

• Sample Preparation Optimization:

  • Increase protein loading (40-80 μg per lane)

  • Add phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4) if phosphorylation affects epitope recognition

  • Test different lysis buffers to improve protein extraction

  • Include reducing agents (5-10 mM DTT) freshly in sample buffer

  • Avoid excessive heating during sample preparation (65°C for 5 minutes instead of boiling)

• Transfer Efficiency Enhancement:

  • Optimize transfer conditions for the molecular weight of SPBC19G7.04

  • Consider semi-dry transfer for potentially faster and more efficient transfer

  • Use transfer membrane with appropriate pore size (0.2 μm PVDF for smaller proteins)

  • Add SDS (0.1%) to transfer buffer if protein is difficult to transfer

  • Stain membrane with Ponceau S to confirm successful protein transfer

• Antibody Incubation Modifications:

  • Increase primary antibody concentration (try 1:250 or 1:100 dilutions)

  • Extend incubation time to 48 hours at 4°C

  • Test different diluents (5% BSA vs. 5% milk)

  • Try different blocking agents (commercial blockers, 5% BSA, or milk)

  • Add 0.1% Tween-20 to antibody diluent to reduce background

• Signal Enhancement Techniques:

  • Use more sensitive detection substrates (enhanced chemiluminescence plus)

  • Consider signal amplification systems (biotin-streptavidin)

  • Increase exposure time during imaging

  • Try different secondary antibodies or higher secondary antibody concentration

  • Consider using a different detection method (fluorescent secondaries)

• Epitope Accessibility Improvement:

  • Include 0.1% SDS in the blocking buffer to enhance epitope exposure

  • Try antigen retrieval techniques adapted from immunohistochemistry

  • Use denatured vs. native conditions systematically

• Storage and Handling Inspection:

  • Check antibody storage conditions and expiration date

  • Minimize freeze-thaw cycles by using small aliquots

  • Ensure all buffers are fresh and correctly prepared

If signal remains problematic, consider performing Western blot using enriched fractions or immunoprecipitated samples to increase target protein concentration relative to total protein load .

How can cross-reactivity issues with SPBC19G7.04 antibody be identified and mitigated in experimental settings?

Identifying and mitigating cross-reactivity issues with SPBC19G7.04 antibody requires a systematic approach:

Identification Strategies:

• Comparative Blotting Analysis:

  • Run parallel Western blots with wild-type and SPBC19G7.04 deletion strains

  • Compare banding patterns to identify non-specific bands present in both samples

  • Analyze multiple S. pombe strains with varying expression levels of the target

• Mass Spectrometry Validation:

  • Excise and identify bands of unexpected molecular weights using LC-MS/MS

  • Compare proteins identified to in silico predictions of potential cross-reactive proteins

• Bioinformatic Analysis:

  • Perform sequence similarity searches to identify S. pombe proteins with similar epitopes

  • Conduct epitope mapping to determine which regions of SPBC19G7.04 are recognized by the antibody

• Peptide Competition Assays:

  • Pre-incubate antibody with excess purified SPBC19G7.04 protein or immunizing peptide

  • Compare results with and without competition to identify specific vs. non-specific signals

Mitigation Strategies:

• Experimental Design Adjustments:

  • Include proper controls in every experiment (deletion strains, pre-immune serum controls)

  • Design experiments that incorporate orthogonal detection methods for validation

• Antibody Purification:

  • Consider affinity purification against the specific antigen

  • Use pre-absorption techniques with lysates from deletion strains to remove cross-reactive antibodies

• Protocol Modifications:

  • Increase stringency of washing steps (higher salt concentration in wash buffers)

  • Adjust blocking conditions (switch from milk to BSA or commercial blockers)

  • Optimize antibody dilution to minimize non-specific binding

  • Reduce incubation time to favor high-affinity specific interactions

• Alternative Detection Strategies:

  • Consider using epitope-tagged versions of SPBC19G7.04 and commercial tag antibodies

  • Develop alternative detection methods such as activity assays or reporter systems

• Data Analysis Considerations:

  • Clearly document and report all bands observed, not just those of expected size

  • Use quantitative analysis that accounts for background and non-specific signals

The polyclonal nature of the SPBC19G7.04 antibody means some degree of cross-reactivity is possible, particularly in a compact genome like S. pombe where related proteins may share sequence similarities. Careful validation and control experiments are essential for confident interpretation of results .

What techniques can improve reproducibility when using different lots of SPBC19G7.04 polyclonal antibody?

Ensuring reproducibility across different lots of polyclonal antibodies like SPBC19G7.04 is a common challenge in research. Implement these strategies to maintain consistent results:

• Standardized Lot Testing Protocol:

  • Develop a standardized validation protocol for each new antibody lot

  • Create a reference lysate batch to use for all lot testing

  • Document lot-specific optimal working dilutions for each application

  • Generate and maintain a checklist of quality control parameters

• Side-by-Side Comparison Analysis:

  • Run parallel experiments with old and new antibody lots

  • Calculate correction factors if signal intensity varies between lots

  • Document and archive representative images from each lot

  • Consider using a standard curve with recombinant protein for quantitative applications

• Internal Controls Implementation:

  • Include consistent positive and negative controls in every experiment

  • Use an internal reference sample across all experiments for normalization

  • Consider spike-in controls of recombinant SPBC19G7.04 at known concentrations

  • Maintain a laboratory reference standard for comparative analysis

• Bulk Purchasing and Storage Strategies:

  • Purchase larger antibody amounts of a single lot for long-term projects

  • Aliquot antibodies into single-use volumes to avoid freeze-thaw cycles

  • Document storage conditions and track time at each storage temperature

  • Consider lyophilization for very long-term storage needs

• Data Normalization Approaches:

  • Develop lot-specific normalization factors based on standard samples

  • Use relative quantification rather than absolute values when comparing across lots

  • Implement statistical methods appropriate for inter-lot variations

  • Consider using ratios to internal controls rather than absolute signals

• Advanced Analytical Methods:

  • Characterize each lot by epitope mapping if resources permit

  • Evaluate lot-specific affinity and avidity through surface plasmon resonance

  • Assess lot-to-lot variations in recognizing post-translationally modified proteins

  • Document specific non-target bands for each lot

By implementing these strategies, researchers can minimize the impact of lot-to-lot variations inherent to polyclonal antibodies like SPBC19G7.04 and maintain experimental reproducibility throughout long-term projects .

How can SPBC19G7.04 antibody be adapted for use in high-throughput screening applications in S. pombe?

Adapting SPBC19G7.04 antibody for high-throughput screening requires methodological innovations to maintain specificity while increasing throughput:

• Automated Western Blot Adaptation:

  • Implement capillary-based automated Western systems (e.g., Jess or Wes platforms)

  • Optimize antibody dilutions specifically for automated systems (typically higher concentrations)

  • Develop standardized lysate preparation protocols compatible with automation

  • Create calibration curves using recombinant standards for quantitative analysis

• Multiplexed Detection Systems:

  • Combine SPBC19G7.04 antibody with antibodies against other targets using spectrally distinct fluorophores

  • Develop multiplexed ELISA arrays for simultaneous detection of multiple proteins

  • Implement bead-based assays (similar to Luminex) for higher sample throughput

  • Validate each antibody separately and in combination to ensure no interference

• Microfluidic Implementations:

  • Adapt antibody for microfluidic chip-based detection systems

  • Determine optimal surface functionalization for antibody immobilization

  • Optimize flow rates and incubation times for on-chip detection

  • Develop image analysis algorithms for automated data interpretation

• Cell-Based High-Content Screening:

  • Optimize fixation and permeabilization conditions for S. pombe cells in 96/384-well formats

  • Develop nuclear and cytoplasmic counterstains compatible with SPBC19G7.04 detection

  • Implement automated image acquisition and analysis pipelines

  • Create scoring algorithms for phenotypic classification based on SPBC19G7.04 localization or expression

• Assay Miniaturization Strategies:

  • Reduce reaction volumes to nanoliter scale using acoustic liquid handling

  • Determine minimum cell number required for reliable detection

  • Establish optimal signal amplification methods for miniaturized formats

  • Validate assay performance metrics (Z' factor, signal-to-background ratio) in reduced volumes

While adapting high-throughput methods, researchers should implement appropriate quality control measures including:

• Plate-based controls (positive, negative, blank)

• Monitoring assay drift across plates and batches

• Implementing robust statistical analysis for hit identification

• Confirming hits with orthogonal lower-throughput methods

These approaches can transform SPBC19G7.04 antibody from a traditional research tool to an enabler of large-scale screening efforts in S. pombe research .

What considerations are important when integrating SPBC19G7.04 antibody-based techniques with other -omics approaches?

Integrating SPBC19G7.04 antibody-based techniques with other -omics approaches requires careful consideration of several factors to ensure compatible and complementary data generation:

• Sample Preparation Harmonization:

  • Develop unified sample preparation protocols that preserve both protein epitopes and other biomolecules (RNA, metabolites)

  • Create fractionation schemes that allow parallel analysis of different biomolecule types

  • Establish sample handling workflows that minimize degradation across all analyte types

  • Consider the impact of fixation and extraction methods on multi-omics compatibility

• Temporal and Spatial Coordination:

  • Implement time-course experiments with synchronized sampling for antibody-based and -omics analyses

  • Develop subcellular fractionation protocols compatible with both approaches

  • Consider single-cell methodologies that allow correlation between protein localization and other -omics data

  • Create unified metadata structures to facilitate cross-platform data integration

• Data Integration Frameworks:

  • Establish normalization strategies to allow comparison across platforms

  • Develop computational pipelines that integrate antibody-based quantification with transcriptomics, proteomics, or metabolomics data

  • Implement machine learning approaches for pattern recognition across multi-omics datasets

  • Create visualization tools that highlight correlations between SPBC19G7.04 detection and other -omics signatures

• Validation Strategies:

  • Design orthogonal validation experiments that verify findings across platforms

  • Implement spike-in standards for cross-platform calibration

  • Develop statistical frameworks for assessing significance in integrated datasets

  • Create benchmark datasets to evaluate integration methodologies

• Technical Compatibility Considerations:

  • Assess buffer compatibilities between antibody-based techniques and -omics sample preparation

  • Evaluate potential interference from detergents, chaotropic agents, or other additives

  • Determine minimum sample input requirements across all platforms

  • Establish quality control metrics applicable across integrated approaches

By carefully considering these factors, researchers can generate more comprehensive and mechanistically insightful data by combining the specificity of SPBC19G7.04 antibody-based detection with the breadth of various -omics approaches .

How might advances in antibody engineering impact future applications of research tools like SPBC19G7.04 antibody?

Emerging advances in antibody engineering are poised to transform research tools like SPBC19G7.04 antibody, creating new capabilities and applications:

• Recombinant Antibody Technology:

  • Conversion of polyclonal antibodies like SPBC19G7.04 to defined recombinant antibody mixtures

  • Cloning of specific high-affinity antibody sequences from polyclonal sera

  • Engineering for improved stability and reduced lot-to-lot variation

  • Creation of renewable antibody sources through expression systems

• Format Diversification:

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

  • Engineering of nanobodies or single-domain antibodies for applications requiring smaller size

  • Creation of bispecific formats combining SPBC19G7.04 recognition with other target specificities

  • Design of intrabodies for live-cell applications and direct functional perturbation

• Functional Modifications:

  • Site-specific conjugation of fluorophores at defined stoichiometry

  • Engineering of antibodies with conditional binding properties (pH-sensitive, temperature-responsive)

  • Development of antibodies with catalytic activities (abzymes) for direct functional studies

  • Creation of split-antibody systems for proximity-based detection

• Improved Specificity Engineering:

  • Epitope-focused selection to improve specificity for particular protein domains

  • Engineering to distinguish between post-translational modification states

  • Development of conformation-specific antibodies for functional studies

  • Computational design to minimize cross-reactivity with related proteins

• Production and Delivery Innovations:

  • In vitro display technologies for rapid antibody generation and evolution

  • Cell-free expression systems for contamination-free antibody production

  • Microfluidic platforms for automated antibody screening and optimization

  • Gene therapy approaches for in situ antibody expression in model systems

These advances will likely transform SPBC19G7.04 from a traditional polyclonal reagent to a precisely engineered research tool with expanded capabilities, including:

• Improved reproducibility through defined composition

• Enhanced functionality through rational engineering

• Expanded application range through format diversification

• Greater specificity through targeted selection and computational design

• Increased accessibility through novel production platforms

Researchers should stay informed about these developments and consider how emerging antibody technologies might enhance their specific applications involving SPBC19G7.04 and other research antibodies .