SPBC800.12c Antibody

What is SPBC800.12c and why is it significant for research?

SPBC800.12c is a protein coding gene in Schizosaccharomyces pombe (fission yeast). While specific information about SPBC800.12c is limited in the provided search results, gene products from the SPBC800 family are known to be involved in translational regulation . Antibodies targeting these proteins are valuable tools for studying gene expression regulation, protein localization, and function in eukaryotic cells. When designing experiments with SPBC800.12c antibodies, researchers should consider the evolutionary conservation of this protein region and potential cross-reactivity with related proteins, similar to considerations made when working with other specific antibody targets .

What are the recommended validation methods for SPBC800.12c antibodies?

Validation of SPBC800.12c antibodies should follow the same rigorous standards applied to other research antibodies. A comprehensive validation approach includes:

• Western blotting to verify molecular weight and specificity

• Immunoprecipitation to confirm target binding

• Immunofluorescence to assess subcellular localization

• Knockout/knockdown controls to confirm antibody specificity

• Cross-reactivity testing against related proteins

These validation steps are critical for ensuring experimental reproducibility. When reporting results, include detailed information about antibody source, catalog number, dilution factors, and validation methods employed .

What expression and purification methods are most effective for SPBC800.12c antibody production?

Based on established antibody production methodologies, the most effective approach for SPBC800.12c antibody production typically involves:

• Cloning the antibody genes into expression vectors

• Transfection and expression in mammalian cell lines such as HEK293T or Expi293F cells

• Purification using affinity chromatography

For mammalian expression systems, transfection should be performed according to standardized protocols, such as using the Expi293 Expression System Kit, with cells maintained at 37°C and 8% CO₂ under shaking conditions (130 rpm) . Purification typically employs MabSelect SURE affinity chromatography followed by size exclusion chromatography to ensure high purity .

How should researchers design experiments to assess SPBC800.12c antibody specificity?

Designing experiments to assess SPBC800.12c antibody specificity requires a multi-faceted approach:

• ELISA-based specificity testing: Coat plates with purified SPBC800.12c protein (2.5-5 μg per well) and related proteins. Block with 3% BSA and perform binding assays with serial dilutions of the antibody. Detection should use appropriate secondary antibodies conjugated to HRP, with results analyzed by luminescence using high-sensitivity substrate .

• Cross-reactivity assessment: Test against related proteins from the same family to determine off-target binding. This can be quantified as a percentage of binding relative to the target protein.

• Epitope mapping: Determine the specific region of SPBC800.12c recognized by the antibody using peptide arrays or truncation mutants.

• Competition assays: Perform with known ligands or binding partners of SPBC800.12c to ensure antibody binding doesn't interfere with functional interactions.

• Control experiments: Include isotype controls and pre-immune serum controls to establish baseline signals.

What cell culture systems are optimal for studying SPBC800.12c function with antibodies?

For studying SPBC800.12c function using antibodies, researchers should consider:

• Fission yeast systems: As SPBC800.12c is a S. pombe gene, native expression studies are optimally conducted in fission yeast. Culturing should follow standard protocols for this organism.

• Mammalian cell expression systems: For heterologous expression, mammalian cell lines such as HEK293T cells can be used. These should be maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 1% Penicillin/Streptomycin, and 1% L-Glutamine at 37°C with 5% CO₂ .

• Specialized expression systems: For high-yield antibody production, Expi293F cells maintained in Expi293 expression medium are recommended .

The selection of an appropriate cell system depends on the specific research question, with consideration given to post-translational modifications, protein folding, and functional interactions that may be species or cell-type specific.

What are the best protocols for using SPBC800.12c antibodies in immunoprecipitation experiments?

For optimal immunoprecipitation with SPBC800.12c antibodies:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Clarify lysate by centrifugation (14,000 × g, 10 minutes, 4°C)

  • Pre-clear with protein A/G beads to reduce non-specific binding

• Antibody binding:

  • Incubate 1-5 μg of antibody with 500 μg of protein lysate

  • Allow binding to occur overnight at 4°C with gentle rotation

• Capture and washing:

  • Add protein A/G beads and incubate for 2-4 hours at 4°C

  • Wash 4-5 times with cold wash buffer (PBS with 0.1% detergent)

  • Elute bound proteins using SDS sample buffer or gentle elution buffer

• Analysis:

  • Analyze by SDS-PAGE followed by western blotting or mass spectrometry

  • Include appropriate controls (isotype control, input sample)

This protocol can be adapted from established methods used for similar antibody-based precipitation experiments .

How can SPBC800.12c antibodies be effectively used in high-throughput screening assays?

For high-throughput applications with SPBC800.12c antibodies:

• Automation-compatible protocols:

  • Adapt antibody-based detection to 384-well plate formats

  • Optimize antibody concentrations to minimize usage while maintaining signal-to-noise ratio

  • Consider using fluorophore-conjugated secondary antibodies for multiplexed detection

• Bead-based assays:

  • Couple SPBC800.12c antibodies to magnetic beads for pull-down assays

  • Use streptavidin-biotin systems for enhanced sensitivity:

    • Biotinylate antibodies using standard NHS-ester chemistry

    • Couple to streptavidin-coated beads or surfaces

    • Validate binding efficiency and specificity before large-scale screening

• Microfluidic systems:

  • Implement microfluidic chips similar to the Bruker Cellular Analysis Beacon system for single-cell analysis

  • Optimize cell loading at approximately 8.0 × 10⁶ cells/mL

  • Use opto-electro positioning (OEP) for precise cell manipulation

• Data analysis:

  • Establish clear positive and negative controls

  • Implement automated image analysis for consistent scoring

  • Use statistical methods appropriate for high-throughput data interpretation

What are the recommended approaches for resolving data inconsistencies when using SPBC800.12c antibodies?

When researchers encounter inconsistent results with SPBC800.12c antibodies, a systematic troubleshooting approach is essential:

• Antibody validation reassessment:

  • Re-validate antibody specificity using western blot and ELISA

  • Test multiple antibody lots if available

  • Consider epitope accessibility in different experimental conditions

• Experimental variables analysis:

  • Create a table of all experimental conditions and outcomes

  • Systematically modify one variable at a time to identify the source of variability

  • Document temperature, pH, buffer composition, and incubation times

• Sample preparation investigation:

  • Evaluate protein extraction methods

  • Assess protein degradation through time-course experiments

  • Consider post-translational modifications that might affect epitope recognition

• Statistical approach:

  • Increase biological and technical replicates

  • Apply appropriate statistical tests to determine significance of variations

  • Consider Bayesian analysis for complex datasets with multiple variables

• Orthogonal method validation:

  • Confirm findings using antibody-independent methods

  • Compare results with genetic approaches (knockout/knockdown)

  • Consider mass spectrometry-based validation for protein identification

How can researchers optimize SPBC800.12c antibody use in multiplexed imaging applications?

For multiplexed imaging with SPBC800.12c antibodies:

• Fluorophore selection and optimization:

  • Choose fluorophores with minimal spectral overlap

  • Consider quantum yield and photostability characteristics

  • Optimize signal-to-noise ratio for each channel

• Sequential staining protocols:

  • Develop validated protocols for antibody stripping and re-probing

  • Establish baseline controls for each staining round

  • Document and account for signal loss during sequential procedures

• Sample preparation considerations:

  • Optimize fixation methods to preserve epitope accessibility

  • Test various antigen retrieval techniques if working with fixed tissue

  • Validate penetration depth in three-dimensional samples

• Image acquisition parameters:

  • Establish consistent exposure settings for quantitative comparison

  • Implement flat-field correction to account for illumination non-uniformity

  • Use appropriate negative controls for autofluorescence subtraction

• Analysis workflow:

  • Develop automated segmentation algorithms for consistent analysis

  • Implement colocalization metrics with statistical validation

  • Consider machine learning approaches for complex pattern recognition

What are the key considerations for using SPBC800.12c antibodies in cryo-EM structural studies?

Implementing SPBC800.12c antibodies in cryo-EM studies requires careful consideration of:

• Antibody fragment preparation:

  • Generate Fab fragments using activated papain (1:100 ratio) at 37°C for one hour

  • Purify using affinity chromatography followed by size exclusion chromatography

  • Confirm homogeneity by analytical SEC and SDS-PAGE

• Complex formation and stability:

  • Optimize antibody:target ratios through titration experiments

  • Assess complex stability using analytical ultracentrifugation

  • Consider crosslinking strategies for stabilizing transient complexes

• Sample vitrification:

  • Optimize buffer conditions to prevent phase separation

  • Test various grid types and surface treatments

  • Implement systematic blotting time optimization

• Data collection strategy:

  • Consider antibody orientation bias in particle distribution

  • Implement tilt series collection if preferred orientations are observed

  • Optimize electron dose to balance resolution and radiation damage

• Data processing considerations:

  • Implement focused classification approaches for heterogeneous samples

  • Consider antibody flexibility in refinement strategies

  • Validate structures using independent antibody fragments or binding partners

How should researchers approach epitope mapping for SPBC800.12c antibodies?

A comprehensive epitope mapping strategy for SPBC800.12c antibodies includes:

• Peptide array analysis:

  • Design overlapping peptides (15-20 amino acids) covering the full SPBC800.12c sequence

  • Include alanine scanning for critical residues

  • Analyze binding patterns to identify continuous epitopes

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare deuterium uptake patterns in free protein versus antibody-bound states

  • Identify regions with reduced exchange rates in the bound state

  • Generate deuterium uptake difference plots to visualize epitope regions

• Mutagenesis approaches:

  • Create targeted mutations in predicted epitope regions

  • Express and purify mutant proteins

  • Assess changes in antibody binding affinity

• Computational prediction and validation:

  • Use structure-based epitope prediction algorithms

  • Validate predictions with experimental approaches

  • Refine models based on experimental results

• X-ray crystallography or cryo-EM:

  • For definitive epitope mapping, solve the structure of antibody-antigen complex

  • Compare experimental structure with computational predictions

  • Identify specific contact residues and structural elements

The epitope information should be documented in standardized formats to facilitate comparison with other antibodies and contribute to community resources.

What quantitative methods are recommended for determining SPBC800.12c antibody binding kinetics?

For precise quantification of SPBC800.12c antibody binding kinetics:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified SPBC800.12c or antibody on sensor chips

  • Determine association (k_on) and dissociation (k_off) rates

  • Calculate equilibrium dissociation constant (K_D) from kinetic parameters

  • Validate at multiple concentrations and flow rates

• Bio-Layer Interferometry (BLI):

  • Use streptavidin sensors for biotinylated protein or antibody

  • Measure real-time binding at different concentrations

  • Perform global fitting to determine kinetic parameters

  • Compare with SPR results for cross-validation

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters (ΔH, ΔS, ΔG)

  • Determine stoichiometry and binding affinity

  • Perform at different temperatures to assess enthalpy-entropy compensation

• Microscale Thermophoresis (MST):

  • Label one binding partner with a fluorescent tag

  • Measure changes in thermophoretic mobility upon binding

  • Determine K_D values across a range of conditions

  • Use for difficult-to-immobilize samples

These quantitative methods should be performed with proper controls and replicated at least three times to ensure reliability of the kinetic parameters.

How does the performance of SPBC800.12c antibodies compare across different experimental platforms?

A systematic comparison of SPBC800.12c antibody performance across platforms should consider:

Researchers should validate antibodies separately for each application and maintain detailed records of optimization parameters for reproducibility.

What are the most effective approaches for troubleshooting non-specific binding issues with SPBC800.12c antibodies?

When encountering non-specific binding with SPBC800.12c antibodies:

• Blocking optimization:

  • Test different blocking agents (BSA, milk, serum, commercial blockers)

  • Optimize blocking time and temperature

  • Consider adding detergents (0.1-0.3% Tween-20) to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform systematic titration experiments

  • Consider brief pre-adsorption with cell/tissue lysates from negative control samples

  • Reduce incubation time while maintaining sufficient signal

• Buffer modification strategies:

  • Adjust salt concentration (150-500 mM NaCl) to reduce ionic interactions

  • Test different pH conditions that maintain antigen-antibody binding

  • Add competitors for common non-specific interactions (0.1-0.5% BSA)

• Secondary antibody considerations:

  • Validate secondary antibody specificity

  • Use highly cross-adsorbed secondary antibodies

  • Consider directly conjugated primary antibodies to eliminate secondary antibody issues

• Sample preparation refinement:

  • Optimize protein extraction methods to reduce interfering components

  • Implement additional purification steps if necessary

  • Address potential post-translational modifications that might affect specificity

How can researchers effectively integrate SPBC800.12c antibody data with other -omics datasets?

For effective integration of antibody-derived data with -omics datasets:

• Data normalization approaches:

  • Implement robust normalization methods appropriate for each data type

  • Consider batch effect correction for datasets generated at different times

  • Validate normalization using control samples or spike-ins

• Correlation analysis strategies:

  • Calculate Pearson or Spearman correlations between antibody-derived measurements and corresponding mRNA levels

  • Develop scatter plots with regression lines to visualize relationships

  • Account for time delays between transcription and translation

• Network analysis methods:

  • Integrate antibody data into protein-protein interaction networks

  • Implement weighted gene co-expression network analysis (WGCNA)

  • Visualize networks using platforms like Cytoscape with antibody-derived data as node attributes

• Machine learning integration:

  • Use supervised learning to identify patterns across multi-omics datasets

  • Implement dimensionality reduction techniques (PCA, t-SNE, UMAP)

  • Validate predictive models using independent datasets

• Pathway enrichment analysis:

  • Map antibody-derived protein measurements to canonical pathways

  • Perform gene set enrichment analysis incorporating protein abundance data

  • Visualize results using pathway mapping tools

These integration approaches should be documented with sufficient detail to allow reproduction by other researchers and should acknowledge the different sources of technical and biological variation inherent in each data type.

What emerging technologies are most promising for enhancing SPBC800.12c antibody research?

Several cutting-edge technologies show particular promise for advancing SPBC800.12c antibody research:

• Single-cell proteomics:

  • Integration with antibody-based detection methods

  • Analysis of heterogeneity in SPBC800.12c expression at single-cell resolution

  • Correlation with transcriptomic data at single-cell level

• Advanced multiplexing approaches:

  • Cyclic immunofluorescence with 20+ antibodies on the same sample

  • Mass cytometry (CyTOF) for high-parameter protein detection

  • DNA-barcoded antibody technologies for ultrahigh-multiplexing

• In situ structural analysis:

  • Proximity ligation assays for protein interaction studies

  • MINFLUX super-resolution microscopy for nanoscale localization

  • Expansion microscopy for improved spatial resolution of protein complexes

• Microfluidic antibody screening:

  • Advanced optofluidic systems similar to the Beacon platform for high-throughput antibody characterization

  • Droplet-based microfluidics for single-cell secretion analysis

  • Integrated systems for rapid antibody-antigen binding kinetics

• AI-assisted antibody engineering:

  • Machine learning for epitope prediction and antibody design

  • Computational modeling of antibody-antigen interfaces

  • Structure-guided antibody optimization

Researchers should consider how these emerging technologies might be applied to address specific questions about SPBC800.12c function and regulation.

What are the key methodological challenges that remain unsolved in SPBC800.12c antibody research?

Despite significant advancements, several methodological challenges persist in SPBC800.12c antibody research:

• Reproducibility across laboratories:

  • Standardization of antibody validation protocols

  • Development of universal reference standards

  • Implementation of robust reporting standards for antibody experiments

• Detection of post-translational modifications:

  • Generation of modification-specific antibodies with high specificity

  • Methods for multiplexed detection of different modification states

  • Quantitative analysis of modification stoichiometry

• Temporal dynamics measurement:

  • Real-time imaging of protein dynamics in living cells

  • Methods for capturing transient interactions or conformational changes

  • Integration of kinetic data with structural information

• Tissue penetration and accessibility:

  • Improved methods for antibody delivery across biological barriers

  • Enhanced protocols for tissue clarification and antibody penetration

  • Strategies for reducing non-specific binding in complex tissues

• Quantitative accuracy:

  • Absolute quantification methods for target proteins

  • Correction for epitope masking effects

  • Standardized approaches for determining limits of detection and quantification

Addressing these challenges will require interdisciplinary approaches combining expertise in biochemistry, engineering, computational biology, and statistics.

How might SPBC800.12c antibody research contribute to understanding broader biological questions?

Research utilizing SPBC800.12c antibodies has potential to advance understanding in several fundamental areas:

• Translational regulation mechanisms:

  • If SPBC800.12c functions similarly to other SPBC800 family members like sum2 (SPBC800.09), it may play roles in translational repression

  • Antibodies could help elucidate its interaction partners and regulatory mechanisms

  • This could provide insights into evolutionarily conserved translational control pathways

• Cell cycle regulation in eukaryotes:

  • Many S. pombe proteins have important roles in cell cycle control

  • Antibody-based studies could reveal cell cycle-dependent localization or modification patterns

  • This may uncover new regulatory mechanisms conserved in higher eukaryotes

• Stress response pathways:

  • Antibodies could be used to track changes in SPBC800.12c levels, modifications, or localization during various stress conditions

  • This might reveal its role in cellular adaptation to environmental challenges

  • Understanding these mechanisms could have broader implications for stress biology

• Evolutionary conservation of protein function:

  • Comparative studies using antibodies recognizing homologous proteins in different species

  • Analysis of functional conservation and divergence across evolutionary time

  • Insights into the evolution of fundamental cellular processes

• Development of new research tools:

  • Methods developed for SPBC800.12c antibody research may be applicable to other challenging protein targets

  • Innovative protocols might become standard approaches in the field

  • Cross-disciplinary collaborations could yield broadly applicable technological advances

These contributions would extend beyond the specific study of SPBC800.12c to impact multiple fields of biological research.