SPBC1773.12 Antibody

What is SPBC1773.12 and why is it significant for S. pombe research?

SPBC1773.12 refers to a specific gene locus in the Schizosaccharomyces pombe genome, with antibodies raised against its protein product being valuable for studying cellular processes in this model organism. S. pombe serves as an excellent model system for investigating eukaryotic cell biology due to its genetic tractability and conservation of fundamental cellular mechanisms. Antibodies targeting SPBC1773.12 enable researchers to track protein localization, expression levels, and interactions in various experimental conditions. These antibodies typically recognize epitopes specific to the SPBC1773.12 protein product, making them valuable for investigating protein function in contexts such as cell cycle progression, stress response, or other cellular processes, similar to applications seen with other S. pombe proteins .

How do I determine the specificity of a SPBC1773.12 antibody?

Determining antibody specificity requires a multi-faceted approach combining several validation techniques. Begin with Western blot analysis using both wild-type and SPBC1773.12 deletion strains to confirm the antibody detects a band of the expected molecular weight that disappears in the knockout. Immunoprecipitation followed by mass spectrometry provides rigorous validation by identifying the pulled-down proteins. Additionally, immunostaining with parallel analysis of GFP-tagged SPBC1773.12 strains helps confirm proper localization patterns. Cross-reactivity assessment against related S. pombe proteins is essential for confirming specificity, particularly when dealing with protein families that may share structural similarities. Documentation of these validation experiments should include positive and negative controls, similar to the validation approaches used for other S. pombe antibodies like SPBC1773.03c .

What sample preparation methods are optimal for SPBC1773.12 antibody applications?

Sample preparation methods vary by application but share core principles for preserving epitope integrity. For Western blot applications, harvest S. pombe cells in mid-log phase (OD600 0.5-0.8) and lyse using either mechanical disruption (glass beads) or enzymatic approaches (zymolyase treatment followed by detergent lysis). Include protease inhibitors (PMSF, leupeptin, pepstatin) and phosphatase inhibitors if phosphorylation status is relevant. For immunofluorescence, fixation with 3-4% paraformaldehyde for 15-30 minutes followed by cell wall digestion with zymolyase (1mg/ml, 30 minutes at 30°C) typically preserves epitope accessibility. Permeabilization with 0.1% Triton X-100 enables antibody penetration while maintaining cellular structure. When performing immunoprecipitation, gentler lysis buffers containing 0.5-1% NP-40 or Triton X-100 help maintain protein-protein interactions. These preparation methods align with established protocols for working with fission yeast antibodies like those in the SPBC series .

How should I optimize Western blot protocols for SPBC1773.12 antibody detection?

Optimization for Western blot detection of SPBC1773.12 requires systematic adjustment of multiple parameters. Begin with antibody titration experiments testing concentrations between 0.5-5 μg/mL (similar to the working range established for antibodies like anti-SP17 ) to determine optimal signal-to-noise ratio. Membrane blocking conditions significantly impact background—test both 5% non-fat milk and 3-5% BSA in TBS-T, as some antibodies perform better with specific blocking agents. For signal development, compare enhanced chemiluminescence (ECL), fluorescent secondary antibodies, and chromogenic detection to determine which provides appropriate sensitivity for your expression levels. Transfer conditions may require optimization—adjust methanol percentage in transfer buffer (10-20%) and transfer time (1-2 hours at 100V or overnight at 30V) based on protein size. Include a loading control antibody (anti-tubulin or anti-actin) for normalization. Document optimal conditions in a standardized protocol:

What controls are essential when using SPBC1773.12 antibody for immunoprecipitation studies?

Rigorous immunoprecipitation experiments require multiple controls to ensure validity and specificity. Always include a negative control using non-specific IgG of the same species and isotype as the SPBC1773.12 antibody to identify non-specific binding. A cellular lysate input control (typically 5-10% of IP material) establishes baseline protein abundance. For definitive specificity validation, perform parallel IPs using: (1) wild-type cells, (2) SPBC1773.12 deletion strains, and (3) strains with epitope-tagged SPBC1773.12. Pre-clearing lysates with protein A/G beads for 1 hour before antibody addition reduces non-specific binding. When investigating protein-protein interactions, validate findings with reciprocal IPs where available antibodies against suspected interaction partners are used to pull down complexes, then probe for SPBC1773.12. Consider crosslinking approaches (such as DSP or formaldehyde) for capturing transient interactions. These comprehensive controls align with best practices for immunoprecipitation experiments across various antibody applications, including those used in therapeutic antibody development .

How can I effectively use SPBC1773.12 antibody for immunofluorescence microscopy in S. pombe?

Successful immunofluorescence with SPBC1773.12 antibody requires protocol optimization focused on preserving both cellular architecture and epitope accessibility. Begin with cell fixation method comparison—test 3.7% formaldehyde (15 minutes), methanol (-20°C, 6 minutes), or a combined approach (formaldehyde followed by methanol) to determine which best preserves epitope recognition while maintaining cellular structure. Cell wall digestion should be carefully calibrated; test zymolyase concentration (0.5-1 mg/ml) and incubation times (15-45 minutes) to achieve balanced cell wall permeabilization without compromising cellular integrity. For antibody incubation, prepare a dilution series (1:100-1:1000) in blocking buffer containing 1-3% BSA and 0.1% Triton X-100 to determine optimal concentration. Counter-staining with DAPI (1 μg/ml) for DNA visualization and phalloidin for actin cytoskeleton provides valuable reference landmarks. Include these essential controls: (1) secondary-only control to assess background, (2) wild-type vs. SPBC1773.12 deletion strains to confirm specificity, and (3) parallel imaging of GFP-tagged SPBC1773.12 strains if available. Similar principles have been successfully applied to immunofluorescence protocols for other antibodies such as those targeting SAP/SLAM-Associated Protein .

How can I adapt SPBC1773.12 antibody for chromatin immunoprecipitation (ChIP) experiments?

Adapting SPBC1773.12 antibody for ChIP requires careful optimization of cross-linking, chromatin fragmentation, and immunoprecipitation conditions. Start by testing cross-linking conditions with 1% formaldehyde for varying durations (5-20 minutes) at room temperature, followed by quenching with 125mM glycine. Sonication parameters must be empirically determined—typically 10-15 cycles of 30 seconds on/30 seconds off at medium intensity—to achieve chromatin fragments of 200-500bp (verify fragment size by agarose gel electrophoresis). For immunoprecipitation, test antibody amounts between 2-10μg per reaction, and consider using a mixture of Protein A and Protein G beads to maximize capture efficiency. ChIP-qPCR validation should target regions with predicted binding sites and control regions without binding sites. For ChIP-seq applications, include an input control and IgG control processed identically to experimental samples. Sequencing library preparation should incorporate size selection (150-300bp) and quality control metrics including complexity assessment and peak distribution analysis. This methodology draws on established ChIP protocols while addressing the specific considerations for nuclear/DNA-associated proteins similar to those studied in various model systems .

What are the considerations for using SPBC1773.12 antibody in quantitative proteomics workflows?

Integrating SPBC1773.12 antibody into quantitative proteomics workflows requires careful planning to ensure accurate protein complex identification and quantification. For immunoprecipitation-mass spectrometry (IP-MS), consider SILAC (Stable Isotope Labeling with Amino acids in Cell culture) labeling of S. pombe cultures—grow control cells in light medium and experimental cells in heavy isotope-labeled medium prior to cell lysis and parallel immunoprecipitation. Mix immunoprecipitated materials at a 1:1 ratio before MS analysis to enable direct quantitative comparison. Alternatively, label-free quantification approaches require highly reproducible immunoprecipitation conditions and multiple biological replicates (minimum n=3). When searching MS data, set appropriate parameters: mass tolerance (10-20 ppm for precursors, 0.02-0.05 Da for fragments), dynamic modifications (oxidation of methionine, acetylation of protein N-terminus), and protein false discovery rate <1%. Significance thresholds for interactors should typically require fold-enrichment >2 and p-value <0.05 compared to controls. Validation of novel interactions should employ orthogonal methods such as co-immunoprecipitation with specific antibodies against identified partners. This approach builds upon established quantitative proteomics methodologies while addressing specific considerations for antibody-based protein complex isolation .

How can I use SPBC1773.12 antibody in conjunction with proximity labeling techniques?

Combining SPBC1773.12 antibody with proximity labeling techniques provides powerful insights into protein interaction networks with spatial and temporal resolution. For antibody-mediated proximity labeling, consider conjugating SPBC1773.12 antibody to enzymes such as APEX2 (engineered ascorbate peroxidase) or TurboID (enhanced biotin ligase) using commercial conjugation kits with NHS-ester chemistry. Optimize conjugation ratios (typically 1:3 to 1:5 molar ratio of antibody:enzyme) and verify activity of the conjugated enzyme through pilot experiments. For cellular applications, membrane permeabilization with digitonin (20-50 μg/ml) enables antibody entry while preserving cellular compartmentalization. After antibody binding (1-2 hours), initiate labeling by adding substrate (biotin-phenol/H₂O₂ for APEX2 or biotin for TurboID) for optimized durations (1 minute for APEX2, 10-30 minutes for TurboID). After cell lysis, capture biotinylated proteins using streptavidin beads and analyze by Western blot or mass spectrometry. Control experiments should include non-specific IgG-enzyme conjugates and unconjugated SPBC1773.12 antibody. This approach builds on emerging proximity labeling technologies while addressing the specific considerations for antibody-directed enzymatic activity .

How can I address non-specific binding issues when using SPBC1773.12 antibody?

Non-specific binding presents a significant challenge in antibody-based applications and requires systematic troubleshooting. Begin by examining blocking conditions—test 5% non-fat milk, 3-5% BSA, 1-5% normal serum from the secondary antibody species, or commercial blocking buffers to identify optimal blocking agents. If background persists, implement additional blocking steps using commercial tools like mouse IgG blocking reagent (if using mouse monoclonals) or avidin/biotin blocking kit (if using biotinylated detection systems). For Western blots, increase washing stringency by adjusting detergent concentration in wash buffers (0.1-0.3% Tween-20 or 0.05-0.1% SDS) and extending wash durations (5-15 minutes per wash, 3-5 washes). Pre-adsorption of the antibody with cell lysate from knockout strains can remove cross-reactive antibodies. For immunofluorescence, autofluorescence can be reduced using sodium borohydride (0.1% for 10 minutes) prior to antibody incubation. Implementation of careful titration experiments often reveals an optimal antibody concentration that maximizes specific signal while minimizing background. Document all optimization steps in a troubleshooting table:

This systematic approach draws from established troubleshooting practices applied to various antibody systems .

How can I verify contradictory results between different detection methods using SPBC1773.12 antibody?

Contradictory results between different detection methods require systematic investigation to resolve discrepancies. Begin by evaluating the antibody itself—confirm it recognizes the same epitope across applications through epitope mapping or competition assays. Different fixation/extraction methods can dramatically affect epitope availability; compare native vs. denatured detection through parallel analysis of the same samples using multiple methods. For discrepancies between Western blot and immunofluorescence, consider that aggregated or misfolded proteins may be detected differently in solution versus in situ. Post-translational modifications can also affect antibody recognition—perform phosphatase treatment or deglycosylation to determine if modifications impact antibody binding. If discrepancies persist, employ orthogonal detection methods such as RNA expression analysis (RT-qPCR), mass spectrometry-based protein identification, or alternative antibodies targeting different epitopes of SPBC1773.12. Integration with genetic approaches—analyzing deletion strains, overexpression constructs, or tagged fusion proteins—can provide definitive validation of antibody specificity. Document all validation experiments in a comparison matrix:

This systematic approach to resolving contradictions draws on methodological validation strategies applied across various antibody systems .

How can SPBC1773.12 antibody be adapted for single-cell protein analysis methods?

Adapting SPBC1773.12 antibody for single-cell protein analysis represents an emerging frontier in S. pombe research. For flow cytometry applications, optimize cell fixation (2-4% paraformaldehyde, 10-20 minutes), permeabilization (0.1-0.3% Triton X-100 or 90% methanol), and antibody concentration (typically 1-5 μg/ml) through titration experiments. Implement fluorescence minus one (FMO) controls and isotype controls to establish gating strategies. For single-cell Western blot approaches, optimize cell settling density (1000-5000 cells/mm²) on poly-lysine coated slides and adjust lysis conditions (typically 0.5-1% SDS with brief heat) to release proteins while maintaining spatial separation. For more advanced techniques like mass cytometry (CyTOF), consider metal conjugation of SPBC1773.12 antibody using commercial conjugation kits with lanthanide metals. Emerging technologies like proximity extension assays (PEA) offer ultrasensitive detection by conjugating paired antibodies with complementary oligonucleotides that enable PCR amplification upon proximal binding. For spatial proteomics applications, consider optimizing SPBC1773.12 antibody for multiplexed ion beam imaging (MIBI) or co-detection by indexing (CODEX) platforms that enable highly multiplexed protein detection in single cells. These approaches build upon emerging single-cell proteomics techniques while addressing the specific considerations for yeast cell analysis and antibody optimization .

What considerations should guide the use of SPBC1773.12 antibody in combination with CRISPR-based genome editing strategies?

Integrating SPBC1773.12 antibody with CRISPR-based genome editing requires careful experimental design for validation and functional characterization. When generating CRISPR knockouts, design at least two guide RNAs targeting different regions of the SPBC1773.12 gene to control for off-target effects. Confirm editing efficiency through genomic PCR and sequencing before using the antibody to verify protein depletion through Western blot analysis. For knock-in applications such as epitope tagging, position tags to minimize interference with antibody epitopes—consider dual validation with both the SPBC1773.12 antibody and tag-specific antibodies to confirm proper expression. When creating point mutations, prioritize mutations outside the antibody epitope region and validate continued antibody recognition before proceeding with functional studies. For temporal control systems like auxin-inducible degron (AID) tagging, establish degradation kinetics through time-course analysis with the SPBC1773.12 antibody, typically sampling at 0, 15, 30, 60, and 120 minutes post-induction. When performing CRISPR activation/repression (CRISPRa/CRISPRi), use the antibody to quantify resulting protein level changes through calibrated Western blotting. These strategies build upon established principles of genome editing validation while addressing the specific considerations for integrating antibody-based protein detection with CRISPR technologies .

How can machine learning approaches enhance data analysis when using SPBC1773.12 antibody in high-content imaging?

Machine learning approaches dramatically enhance the analysis capabilities for high-content imaging data generated using SPBC1773.12 antibody. For image segmentation, train convolutional neural networks (CNNs) using manually annotated training sets (typically 100-200 annotated cells) to accurately identify cell boundaries, nuclei, and subcellular compartments in S. pombe. Feature extraction algorithms can then quantify multiple parameters including signal intensity, texture features, morphological characteristics, and spatial distribution patterns of SPBC1773.12 staining. Implement dimensionality reduction techniques such as principal component analysis (PCA) or t-distributed stochastic neighbor embedding (t-SNE) to visualize complex multi-parameter data in two dimensions. For phenotypic classification, train supervised machine learning models (random forests, support vector machines) using positive and negative control conditions to automatically categorize cells based on SPBC1773.12 staining patterns. Unsupervised clustering approaches can identify novel phenotypic classes without prior annotation. For time-lapse imaging, recurrent neural networks (RNNs) or long short-term memory networks (LSTMs) can track temporal changes in SPBC1773.12 localization or expression. Consider these key performance metrics when implementing machine learning workflows:

This approach builds upon emerging applications of machine learning in biological image analysis while addressing the specific considerations for yeast cell morphology and antibody-based protein detection .