SPAC23H3.15c Antibody

What is SPAC23H3.15c and why is it significant for research?

SPAC23H3.15c is a Schizosaccharomyces pombe-specific protein that has been identified as having important cellular localizations in both the cytosol and nucleus, as demonstrated by direct assay (IDA) evidence . While its GO Process and GO Function annotations remain currently uncharacterized (0 annotations), its presence in these critical cellular compartments suggests potential roles in nuclear transport, signaling, or gene regulation. The protein is significant for research in S. pombe models as it represents a species-specific component that might reveal unique aspects of fission yeast biology .

How do I confirm the specificity of a SPAC23H3.15c antibody?

To confirm antibody specificity for SPAC23H3.15c, implement a multi-validation approach:

• Western blot analysis: Using wild-type S. pombe lysate alongside a SPAC23H3.15c deletion strain. Specific antibodies should show a single band at the predicted molecular weight in wild-type cells and no band in deletion strains.

• Immunoprecipitation followed by mass spectrometry: This approach confirms that the antibody is pulling down the correct target protein.

• Immunofluorescence microscopy: Compare localization patterns against known data. SPAC23H3.15c should show both cytosolic and nuclear localization patterns based on GO annotation data .

• Cross-reactivity testing: Test the antibody against related proteins in S. pombe to ensure it doesn't recognize homologous proteins.

Similar validation approaches have been used successfully for other antibodies as demonstrated in antibody optimization studies .

What cellular compartments does SPAC23H3.15c localize to?

Based on Gene Ontology Cellular Component data, SPAC23H3.15c localizes to:

• Cytosol - Established through direct assay (IDA) evidence

• Nucleus - Also confirmed through direct assay (IDA) evidence

This dual localization pattern suggests the protein may shuttle between these compartments or function in processes that span both cellular regions. When using antibodies for localization studies, consider these known compartments as positive controls for immunofluorescence experiments. The nuclear-cytosolic distribution may vary depending on cell cycle stage or environmental conditions, which should be factored into experimental design.

How can I optimize an antibody against SPAC23H3.15c for improved specificity and sensitivity?

Optimizing antibodies against SPAC23H3.15c can be approached using both computational and experimental strategies:

Computational optimization:

• Implement deep learning frameworks similar to those used for therapeutic antibodies to predict complementarity-determining region (CDR) modifications that could enhance binding affinity and specificity .

• Use geometric neural network models to extract interresidue interaction features and predict changes in binding affinity that would result from amino acid substitutions .

• Simulate an in silico ensemble of predicted complex structures with CDR mutations to obtain robust estimations of free energy changes (ΔΔG) .

Experimental optimization:

• Develop structure-focused antibody libraries displayed on yeast surface as done for therapeutic antibodies .

• Use high-throughput biophysical profiling to screen for variants with improved properties .

• Perform iterative rounds of selection with increasingly stringent conditions, testing single mutations first, then combining beneficial mutations (as seen in the optimization pathway: single → double → triple → quadruple mutations) .

Antibodies enhanced through this approach have shown 20- to 50-fold stronger binding affinities and improved off-rate values, decreasing from 10^-2 to 10^-3, which signifies longer binding periods and higher stability .

What are the technical challenges in detecting SPAC23H3.15c protein interactions using antibody-based approaches?

Detecting protein interactions for SPAC23H3.15c presents several technical challenges:

• Limited known interactors: BioGRID data indicates only 3 known interactors and 3 total interactions for SPAC23H3.15c , suggesting either limited interaction networks or insufficient research to date.

• Cross-reactivity with related proteins: S. pombe contains numerous related proteins that might share epitopes with SPAC23H3.15c.

• Transient interactions: If SPAC23H3.15c forms transient complexes, standard co-immunoprecipitation approaches might fail to capture these interactions.

• Conformational epitopes: If antibodies recognize conformational epitopes, protein denaturation during sample preparation may abolish antibody recognition.

• Epitope masking: When SPAC23H3.15c interacts with other proteins, the epitope recognized by the antibody might become inaccessible.

To overcome these challenges:

• Use in situ proximity ligation assays to detect transient interactions

• Implement chemical crosslinking prior to immunoprecipitation

• Develop antibodies against multiple epitopes of SPAC23H3.15c

• Consider a structured-guided antibody design approach similar to that used for therapeutic antibodies

How do post-translational modifications of SPAC23H3.15c affect antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of SPAC23H3.15c:

• Epitope masking: PTMs such as phosphorylation, acetylation, or ubiquitination can directly block antibody binding sites.

• Conformational changes: PTMs can induce structural changes in SPAC23H3.15c that alter epitope accessibility or conformation.

• Experimental considerations:

  • Generate modification-specific antibodies using synthetic peptides containing the modified residue

  • Use paired antibodies (modification-specific and pan-specific) to determine the proportion of modified protein

  • Implement dephosphorylation treatments prior to immunoblotting to confirm phosphorylation-dependent epitope recognition

  • Analyze samples under conditions that preserve or remove specific modifications

• Validation approach: Compare antibody reactivity against native SPAC23H3.15c versus protein treated with modification-removing enzymes (phosphatases, deacetylases, etc.).

When characterizing SPAC23H3.15c antibodies, it's essential to determine whether they recognize the protein regardless of modification state or are sensitive to specific modifications. This distinction is crucial for experimental interpretation, especially when studying how cellular conditions affect SPAC23H3.15c function.

What are the optimal fixation and permeabilization methods for immunofluorescence detection of SPAC23H3.15c?

Given SPAC23H3.15c's dual localization in both cytosol and nucleus , fixation and permeabilization protocols require careful optimization:

Recommended fixation methods (comparative efficacy):

Permeabilization considerations:

• For paraformaldehyde fixation: 0.1% Triton X-100 (10 min) provides balanced access to both nuclear and cytosolic compartments

• For membrane-associated fractions: Digitonin (50 μg/ml, 5 min) offers gentler permeabilization

• Saponin (0.1%, 10 min): Reversible permeabilization that maintains protein complexes

The dual localization of SPAC23H3.15c necessitates careful balancing of fixation and permeabilization to preserve epitopes while allowing antibody access to both compartments. Testing multiple conditions in parallel is recommended for new antibodies against this protein.

How can I quantitatively assess SPAC23H3.15c expression levels across different experimental conditions?

Quantitative assessment of SPAC23H3.15c expression requires multi-method validation:

• Western blot quantification:

  • Use recombinant SPAC23H3.15c protein to create a standard curve

  • Implement LI-COR near-infrared fluorescence detection for wider linear range

  • Include loading controls appropriate for your experimental conditions (Cdc2 for cell cycle studies, GAPDH for general expression)

• Quantitative microscopy:

  • Measure nuclear/cytoplasmic ratio of SPAC23H3.15c under different conditions

  • Apply automated image analysis workflows to eliminate observer bias

  • Include calibration standards in each imaging session

• qPCR for transcript levels:

  • Design primers specific to SPAC23H3.15c mRNA

  • Validate primer efficiency using standard curves

  • Use multiple reference genes for normalization

• Absolute quantification approaches:

  • Selected Reaction Monitoring (SRM) mass spectrometry with isotope-labeled standards

  • Enzyme-Linked Immunosorbent Assay (ELISA) with purified standards

When analyzing changes in SPAC23H3.15c expression, consider its interactions with other proteins (currently known to have 3 interactors ) as these may influence detection efficiency or stability of the protein.

What controls should be included when using SPAC23H3.15c antibodies for chromatin immunoprecipitation (ChIP) experiments?

Given SPAC23H3.15c's nuclear localization , ChIP experiments require rigorous controls:

Essential controls for SPAC23H3.15c ChIP experiments:

• Input control: Unprocessed chromatin sample to normalize for DNA abundance variations.

• No-antibody control: Complete ChIP procedure without adding the SPAC23H3.15c antibody to identify non-specific binding to beads or matrix.

• Isotype control: Use of a matched isotype antibody of irrelevant specificity to control for non-specific binding.

• Positive control regions: If previous studies have identified SPAC23H3.15c binding sites, include primers for these regions.

• Negative control regions: Include genomic regions unlikely to be bound by SPAC23H3.15c (e.g., heterochromatic regions or genes known to be inactive in your conditions).

• Biological validation controls:

  • ChIP in SPAC23H3.15c deletion or knockdown strains

  • ChIP using two different antibodies against SPAC23H3.15c

  • ChIP under conditions known to affect SPAC23H3.15c localization

• Spike-in normalization: Consider using exogenous chromatin (e.g., from another species) as a normalization control for experiments comparing different conditions.

How can I develop a high-throughput screening assay using SPAC23H3.15c antibodies?

Developing high-throughput screening (HTS) assays with SPAC23H3.15c antibodies can leverage approaches similar to those used in therapeutic antibody development :

Assay development strategy:

• Antibody immobilization formats:

  • Conjugate antibodies to high-capacity plates or beads

  • Consider oriented immobilization using Protein A/G or site-specific biotinylation

  • Test multiple surface chemistries for optimal signal-to-noise ratios

• Detection systems:

  • Develop sandwich ELISA using two non-competing anti-SPAC23H3.15c antibodies

  • Implement homogeneous time-resolved fluorescence (HTRF) for improved throughput

  • Consider AlphaScreen technology for detecting SPAC23H3.15c interactions with known partners

• Assay validation parameters:

  • Z' factor >0.5 indicates excellent assay quality

  • Signal-to-background ratio >10 for robust detection

  • Coefficient of variation <15% for reproducibility

• Miniaturization strategy:

  • Transition from 96-well to 384- or 1536-well formats progressively

  • Adjust antibody concentrations to maintain signal while reducing consumption

  • Implement acoustic liquid handling for nanoliter-scale dispensing

• Biophysical screening methods:

  • Adapt thermal challenge assays similar to those used in antibody engineering

  • Develop label-free detection systems using surface plasmon resonance in array format

  • Consider microfluidic approaches for antibody-antigen interaction analysis

By combining these approaches, researchers can develop robust HTS assays for identifying compounds or conditions that affect SPAC23H3.15c levels, localization, or interactions with its three known binding partners .

What are the best approaches for multiplexed detection of SPAC23H3.15c along with its interaction partners?

For multiplexed detection of SPAC23H3.15c and its interaction partners (of which three are known ), several advanced methodologies are applicable:

Multiplexed detection strategies:

• Multiplexed immunofluorescence:

  • Use spectrally distinct fluorophores for each target protein

  • Implement tyramide signal amplification for enhanced sensitivity

  • Consider sequential antibody labeling and stripping for more than 4-5 targets

  • Analyze colocalization quantitatively using Pearson's or Manders' coefficients

• Mass cytometry (CyTOF):

  • Label antibodies with distinct metal isotopes instead of fluorophores

  • Allows simultaneous detection of 40+ proteins without spectral overlap

  • Particularly useful for analyzing cellular heterogeneity in response to perturbations

• Proximity-based detection methods:

  • Proximity Ligation Assay (PLA) for detecting SPAC23H3.15c interactions in situ

  • FRET/BRET approaches using antibody fragments conjugated to donor/acceptor pairs

  • Enzyme complementation assays (e.g., split HRP or β-lactamase)

• Multiplex Western blotting:

  • Sequential probing with different primary antibodies

  • Multiplex fluorescent detection with spectrally distinct secondary antibodies

  • Consider automated Western systems with internal lane normalization

• Co-immunoprecipitation coupled to multiplexed proteomic analysis:

  • IP with anti-SPAC23H3.15c followed by TMT-labeled mass spectrometry

  • Sequential immunoprecipitation to confirm complex formation

  • Comparison of interaction profiles under different cellular conditions

When designing multiplexed experiments, consider the steric hindrance between antibodies and prioritize validation of antibody combinations to ensure one antibody does not interfere with binding of another.

How can surface plasmon resonance (SPR) be used to characterize SPAC23H3.15c antibody binding kinetics?

Surface plasmon resonance provides valuable insights into antibody-antigen binding kinetics, which can be applied to SPAC23H3.15c antibody characterization:

SPR experimental workflow:

• Surface preparation options:

  • Immobilize purified SPAC23H3.15c protein on CM5 or similar sensor chips via amine coupling

  • Alternatively, capture biotinylated SPAC23H3.15c on streptavidin surfaces

  • For epitope binning, immobilize the antibody and use SPAC23H3.15c as analyte

• Kinetic parameter determination:

  • Measure association rate constant (ka) and dissociation rate constant (kd)

  • Calculate equilibrium dissociation constant (KD = kd/ka)

  • Compare with optimized therapeutic antibodies which show KD values in the 0.42-1.2 nM range and kd values around 10^-3

• Experimental design considerations:

  • Use a concentration series spanning 0.1-10x the expected KD

  • Include buffer-only injections for reference subtraction

  • Implement double-referencing for optimal baseline stability

  • Consider mass transport limitations when analyzing data

• Advanced applications:

  • Epitope binning to map distinct epitopes on SPAC23H3.15c

  • Competition assays to determine if antibodies compete with natural binding partners

  • Temperature dependence studies to evaluate enthalpic and entropic contributions

• Data analysis approach:

  • Fit to appropriate binding models (1:1, heterogeneous ligand, etc.)

  • Evaluate quality of fit using residual plots and chi-square values

  • Compare results across multiple antibody batches for consistency

The SPR approach would be particularly valuable for comparing different antibodies against SPAC23H3.15c and for determining whether an antibody might interfere with the protein's interactions with its three known binding partners .

How do I troubleshoot non-specific binding when using SPAC23H3.15c antibodies in different applications?

Non-specific binding can significantly impact SPAC23H3.15c antibody experiments. Here's a systematic troubleshooting approach:

Western blotting non-specific binding:

Immunofluorescence troubleshooting:

• Pre-absorb antibody with acetone powder from knockout strain

• Include peptide competition controls

• Optimize primary antibody concentration using dilution series

• Evaluate different secondary antibodies for lowest background

• For nuclear staining, treat with DNase to distinguish DNA-associated signals

Immunoprecipitation optimization:

• Pre-clear lysates with beads alone before adding antibody

• Cross-test multiple lysis buffers to preserve epitope while extracting protein

• Consider covalent antibody coupling to beads to eliminate heavy chain signals

• Incorporate stringent wash steps with increasing salt concentration

Advanced approaches should include techniques from therapeutic antibody optimization, such as yeast display, structured-guided antibody design, and library-scale thermal challenge assays that have proven successful in optimizing antibody specificity and stability .

How can I accurately interpret changes in SPAC23H3.15c localization between cytoplasmic and nuclear compartments?

Given SPAC23H3.15c's documented localization in both cytosol and nucleus , interpreting changes in its distribution requires careful experimental design and quantitative analysis:

Quantitative analysis approaches:

• Nuclear/cytoplasmic ratio calculation:

  • Define nuclear regions using DNA staining (DAPI/Hoechst)

  • Calculate signal intensity ratio between nuclear and cytoplasmic compartments

  • Use >30 cells per condition for statistical robustness

  • Apply appropriate statistical tests (e.g., Mann-Whitney for non-parametric data)

• Subcellular fractionation validation:

  • Perform biochemical fractionation to isolate nuclear and cytoplasmic fractions

  • Verify purity using compartment-specific markers (e.g., GAPDH for cytoplasm, Histone H3 for nucleus)

  • Quantify SPAC23H3.15c in each fraction by immunoblotting

• Live-cell imaging considerations:

  • Generate fluorescently-tagged SPAC23H3.15c ensuring tag doesn't disrupt localization

  • Track individual cells over time to capture dynamic shuttling

  • Use photobleaching approaches (FRAP/FLIP) to measure transport rates

• Interpretation guidelines:

  • Consider cell cycle dependency (synchronized cultures may reveal patterns)

  • Evaluate stress responses that might trigger relocalization

  • Account for potential masking of epitopes in specific compartments

  • Always normalize to total protein levels when comparing conditions

How might deep learning approaches be applied to improve SPAC23H3.15c antibody design and optimization?

Deep learning approaches could revolutionize SPAC23H3.15c antibody development, similar to antibody optimization methods used for therapeutic targets :

Potential deep learning applications:

• Epitope prediction and optimization:

  • Train geometric neural networks on antibody-antigen complex structures to predict optimal binding sites on SPAC23H3.15c

  • Identify conserved regions of SPAC23H3.15c that would make stable epitopes

  • Predict how mutations in complementarity-determining regions (CDRs) would affect binding affinity

• Stability enhancement:

  • Apply deep learning frameworks to predict CDR modifications that improve thermal stability

  • Implement in silico ensemble modeling to obtain robust free energy change (ΔΔG) estimations

  • Optimize antibody framework regions for improved expression and reduced aggregation

• Cross-reactivity minimization:

  • Train neural networks on homologous proteins to identify SPAC23H3.15c-specific epitopes

  • Predict potential off-target binding and redesign antibody sequences to enhance specificity

  • Generate synthetic training data augmenting limited experimental datasets

• Implementation strategy:

  • Begin with single mutations to establish baseline improvements, then progress to double, triple, and quadruple mutations as demonstrated in therapeutic antibody optimization

  • Combine computational predictions with high-throughput experimental validation

  • Implement iterative cycles of model improvement based on experimental results

These approaches could significantly accelerate SPAC23H3.15c antibody development, potentially achieving 20- to 50-fold improvements in binding affinity and enhanced off-rate characteristics similar to those observed in therapeutic antibody optimization .

What emerging technologies might enhance the specificity and sensitivity of SPAC23H3.15c detection in complex samples?

Several cutting-edge technologies show promise for enhancing SPAC23H3.15c detection:

Emerging technological approaches:

• Single-molecule detection methods:

  • Single-molecule pull-down (SiMPull) for detecting individual SPAC23H3.15c molecules

  • Total Internal Reflection Fluorescence (TIRF) microscopy for improved signal-to-noise ratio

  • Super-resolution microscopy (STORM/PALM) for precise subcellular localization beyond the diffraction limit

• Nanobody and alternative binding protein technologies:

  • Develop camelid nanobodies against SPAC23H3.15c for improved tissue penetration

  • Engineer non-antibody scaffolds (DARPins, Affibodies) for specific SPAC23H3.15c binding

  • Employ bispecific constructs targeting SPAC23H3.15c and one of its interaction partners simultaneously

• Microfluidic and digital approaches:

  • Digital ELISA platforms for single-molecule detection in dilute samples

  • Droplet microfluidics for high-throughput screening of antibody variants

  • Microfluidic diffusional sizing to measure SPAC23H3.15c-antibody interactions in solution

• CRISPR-based detection systems:

  • Integrate antibody recognition with CRISPR-Cas readouts for signal amplification

  • Develop epitope-tagging strategies compatible with CRISPR knock-in for endogenous detection

  • Apply base editors to introduce epitope tags at the endogenous SPAC23H3.15c locus

• Aptamer-based detection alternatives:

  • Develop DNA/RNA aptamers against SPAC23H3.15c as antibody alternatives

  • Combine aptamers with antibodies in sandwich formats for improved specificity

  • Create aptamer beacons that fluoresce upon SPAC23H3.15c binding

These technologies could overcome current limitations in detecting SPAC23H3.15c, particularly when studying its interactions with its three known binding partners or when analyzing its distribution between nuclear and cytosolic compartments .