SPBC1289.13c Antibody

What is SPBC1289.13c and why is it studied in Schizosaccharomyces pombe research?

SPBC1289.13c is a specific gene/protein in Schizosaccharomyces pombe (fission yeast), an important model organism used extensively in molecular and cellular biology research. This protein has been identified through genome sequencing efforts of S. pombe, which has a relatively small genome of approximately 13.8 Mb distributed across three chromosomes . The comprehensive genome project for S. pombe, completed in 2002, enabled researchers to systematically study individual genes like SPBC1289.13c within the context of cellular processes and regulatory networks . Within fission yeast research, SPBC1289.13c is of interest because of its potential role in cellular processes that are conserved among eukaryotes, making it valuable for translational research into higher organisms. Research on such proteins is particularly valuable because S. pombe has one of the smallest gene complements among free-living eukaryotes, with approximately 5,100 total genes, making it an excellent reductionist model system .

What are the basic properties of the SPBC1289.13c antibody?

The SPBC1289.13c antibody (product code CSB-PA528479XA01SXV) is a polyclonal antibody raised in rabbits against recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) SPBC1289.13c protein . It is supplied in liquid form with a storage buffer consisting of 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . The antibody has been purified using an antigen affinity purification method, which enhances its specificity for the target protein . It is specifically reactive with Schizosaccharomyces pombe (strain 972/ATCC 24843) and has been tested for applications including ELISA and Western Blot (WB) . As with many research antibodies, it is intended exclusively for research use and not for diagnostic or therapeutic applications . Understanding these basic properties is essential for researchers planning experiments involving detection or quantification of SPBC1289.13c in fission yeast samples.

How should researchers optimize Western blot protocols for SPBC1289.13c detection?

For optimal Western blot detection of SPBC1289.13c, researchers should first carefully prepare fission yeast lysates using methods that preserve protein integrity while efficiently extracting the target protein . Cell disruption using glass beads in an appropriate lysis buffer containing protease inhibitors is recommended, with samples being kept cold throughout processing to prevent protein degradation. For protein separation, 10-12% SDS-PAGE gels typically provide appropriate resolution for SPBC1289.13c, though the exact percentage may need adjustment based on the protein's molecular weight . During transfer to membranes, PVDF membranes are often preferred over nitrocellulose for their higher protein binding capacity and durability during stripping/reprobing processes. Blocking should be performed with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature, with primary antibody dilutions typically starting at 1:1000 and optimized based on signal-to-noise ratio in preliminary experiments . For enhanced specificity, overnight primary antibody incubation at 4°C is recommended, followed by extensive washing in TBST to remove unbound antibodies. The secondary antibody selection should match the primary antibody host species (rabbit in this case), and chemiluminescent detection systems generally provide good sensitivity for this application .

What protocols are recommended for immunoprecipitation (IP) using SPBC1289.13c antibody?

For immunoprecipitation of SPBC1289.13c from fission yeast extracts, researchers should begin with a gentle cell lysis procedure to maintain protein-protein interactions, typically using non-ionic detergents such as NP-40 or Triton X-100 at concentrations of 0.5-1% . Prior to the actual IP procedure, it is advisable to perform a pre-clearing step using an irrelevant antibody of the same isotype (IgG) and protein A/G beads to reduce non-specific binding . For the immunoprecipitation itself, typically 2-5 μg of SPBC1289.13c antibody is used per 500 μg of total protein, with overnight incubation at 4°C under gentle rotation to maximize antigen-antibody interaction while preserving complex stability . Protein A or Protein G beads (or a combination) should be added following antibody incubation, with Protein A having stronger affinity for rabbit IgG, making it particularly suitable for this rabbit-derived polyclonal antibody . Washing steps should be carefully optimized; typically, four washes with decreasing salt concentrations help remove non-specifically bound proteins while preserving specific interactions. Elution can be performed using low pH glycine buffer or by boiling in SDS-PAGE sample buffer, with the latter being more complete but potentially more disruptive to the antibody structure for subsequent analyses . For confirmation of successful immunoprecipitation, Western blotting of input, unbound, and eluted fractions is recommended.

How can the SPBC1289.13c antibody be effectively used in immunofluorescence microscopy?

For successful immunofluorescence microscopy with SPBC1289.13c antibody, proper cell fixation and permeabilization are critical first steps . For fission yeast cells, a combination of 3.7% formaldehyde fixation for 30 minutes followed by cell wall digestion with Zymolyase or Lysing Enzymes creates spheroplasts that allow antibody access to intracellular antigens. Cell permeabilization can be achieved using 0.1% Triton X-100, with the exact concentration and duration requiring optimization to balance antigen access with preservation of cellular structures . Blocking with 5% BSA or normal serum from the same species as the secondary antibody helps reduce non-specific binding, improving signal-to-noise ratio in the final images. Primary antibody dilutions typically start at 1:100, though optimal concentrations should be determined empirically through titration experiments . For fluorescent detection, secondary antibodies conjugated to bright, photostable fluorophores like Alexa Fluor dyes are recommended, with appropriate controls including secondary-only samples to assess background fluorescence levels . Counterstaining with DAPI allows visualization of nuclei, providing a reference point for localizing SPBC1289.13c within cellular compartments. Modern confocal microscopy with z-stack acquisition is ideal for determining the precise subcellular localization of SPBC1289.13c in three dimensions, particularly if co-localization with known cellular markers is being investigated .

What are the recommended protocols for ELISA using the SPBC1289.13c antibody?

For ELISA applications with SPBC1289.13c antibody, researchers should first determine whether a direct, indirect, sandwich, or competitive ELISA format is most appropriate for their specific research question . For a standard indirect ELISA, high-binding 96-well plates should be coated with purified recombinant SPBC1289.13c protein at concentrations ranging from 1-10 μg/ml in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C . After washing with PBS-T (PBS containing 0.05% Tween-20), blocking with 2-5% BSA or non-fat dry milk in PBS-T for 1-2 hours at room temperature helps minimize non-specific binding. Serial dilutions of the primary SPBC1289.13c antibody should be prepared (typically starting at 1:500) to determine optimal working concentration, with incubation times of 1-2 hours at room temperature or overnight at 4°C for maximum sensitivity . An HRP-conjugated anti-rabbit secondary antibody is then applied at the manufacturer's recommended dilution (typically 1:2000-1:5000), followed by thorough washing to remove unbound antibody . Signal development using TMB substrate and stopping with 2N H₂SO₄ provides a colorimetric readout that can be quantified at 450 nm using a microplate reader. Standard curves using known concentrations of recombinant SPBC1289.13c protein should be included in each assay for quantitative analysis, along with appropriate negative controls to establish assay specificity .

How can SPBC1289.13c antibody be used to study transcriptional-regulatory networks in fission yeast?

The SPBC1289.13c antibody can serve as a powerful tool for investigating transcriptional-regulatory networks in Schizosaccharomyces pombe, particularly if SPBC1289.13c protein is involved in gene regulation processes . Researchers can employ chromatin immunoprecipitation followed by sequencing (ChIP-seq) using this antibody to identify genomic regions bound by SPBC1289.13c, providing insights into potential target genes under its regulatory control . This approach can be particularly valuable when integrated with transcriptomic data from RNA-seq experiments comparing wild-type and SPBC1289.13c deletion or overexpression strains to correlate binding events with gene expression changes . For mechanistic studies, the antibody can be used in co-immunoprecipitation experiments to identify interacting protein partners within transcriptional complexes, revealing how SPBC1289.13c functions within larger regulatory networks . Researchers studying flocculation in S. pombe, which is governed by complex transcriptional-regulatory networks involving multiple transcription factors (such as Rfl1, Adn2, Adn3, Sre2, Yox1, Mbx2, Cbf11, and Cbf12), might find this antibody particularly useful if SPBC1289.13c plays a role in these processes . When combined with techniques like immunofluorescence microscopy, the antibody can also provide spatial information about SPBC1289.13c localization during different transcriptional states or cell cycle phases, contributing to our understanding of dynamic regulatory processes in fission yeast .

What experimental approaches can be used to validate SPBC1289.13c antibody specificity in fission yeast strains?

Rigorous validation of SPBC1289.13c antibody specificity is essential for ensuring reliable experimental results in fission yeast research. One definitive approach involves comparing Western blot signals between wild-type S. pombe and a SPBC1289.13c gene deletion strain (if viable), as absence of signal in the deletion strain would strongly confirm antibody specificity . For essential genes where deletion may not be possible, researchers can utilize strains with reduced SPBC1289.13c expression through techniques like auxin-inducible degron systems or repressible promoters, expecting proportional reduction in antibody signal intensity . Peptide competition assays provide another validation method, where pre-incubation of the antibody with excess purified recombinant SPBC1289.13c protein or the immunizing peptide should abolish or significantly reduce specific signal in subsequent applications . Cross-reactivity testing against recombinant SPBC1289.13c protein alongside closely related proteins can assess potential recognition of homologous proteins, particularly important in studies examining protein families . For more advanced validation, mass spectrometry analysis of immunoprecipitated material can confirm the identity of proteins recognized by the antibody, providing an independent verification method . Additionally, researchers should perform side-by-side testing of different antibody lots when available, as lot-to-lot variation can occur with polyclonal antibodies like the SPBC1289.13c antibody .

How can researchers integrate SPBC1289.13c antibody data with genomic and proteomic datasets?

Integration of SPBC1289.13c antibody-derived data with genomic and proteomic datasets enables comprehensive understanding of this protein's function within broader cellular contexts. Researchers can combine ChIP-seq data obtained using the SPBC1289.13c antibody with RNA-seq transcriptome profiles to correlate protein binding sites with gene expression changes, identifying direct versus indirect regulatory effects . Quantitative Western blot or ELISA measurements of SPBC1289.13c protein levels across different conditions can be integrated with corresponding mRNA expression data to investigate post-transcriptional regulation mechanisms, including potential discordance between transcript and protein abundance . Co-immunoprecipitation followed by mass spectrometry (IP-MS) using the SPBC1289.13c antibody generates protein interaction networks that can be mapped onto existing protein-protein interaction databases for S. pombe, revealing novel connections or confirming predicted interactions . For spatial studies, immunofluorescence data showing SPBC1289.13c subcellular localization can be integrated with high-throughput organelle proteomics datasets to validate compartment-specific functions . Advanced bioinformatics approaches like Gene Ontology enrichment analysis of SPBC1289.13c-associated genes or proteins can uncover functional patterns that may not be apparent from individual experiments . These integrated analyses are particularly powerful when performed across different environmental conditions or genetic backgrounds, potentially revealing condition-specific functions of SPBC1289.13c within the compact genome of fission yeast .

What are common causes of non-specific binding when using SPBC1289.13c antibody, and how can they be addressed?

Non-specific binding is a frequent challenge when working with polyclonal antibodies like the SPBC1289.13c antibody, often manifesting as multiple bands in Western blots or diffuse signals in immunofluorescence . Insufficient blocking is a primary cause that can be addressed by increasing blocking agent concentration (from 3% to 5% BSA or milk) or extending blocking time from 1 to 2 hours at room temperature . Using antibody diluent containing the blocking agent and 0.1-0.3% Tween-20 can further reduce non-specific interactions during primary and secondary antibody incubations . Overly concentrated primary antibody typically leads to increased background; researchers should perform titration experiments starting with higher dilutions (1:1000) and gradually decreasing if necessary, rather than beginning with concentrated antibody solutions . Cross-reactivity with related proteins in S. pombe can be particularly challenging, especially if SPBC1289.13c belongs to a conserved protein family; pre-absorption of the antibody with recombinant proteins of close homologs or with lysates from strains overexpressing these homologs can enhance specificity . For immunoprecipitation experiments, more stringent washing conditions (increased salt concentration up to 500 mM NaCl or addition of 0.1% SDS to wash buffers) can reduce non-specific binding, though researchers must balance this against the risk of disrupting legitimate weak interactions . If high background persists despite optimization, alternative detection methods like fluorescent secondary antibodies instead of chemiluminescence for Western blots may provide better signal-to-noise ratios in some applications .

How can researchers interpret conflicting results between antibody-based detection and other methods for SPBC1289.13c?

When confronted with discrepancies between antibody-based detection of SPBC1289.13c and alternative methods such as RNA expression data or tagged protein systems, researchers should systematically evaluate several potential explanations. Post-transcriptional regulation mechanisms, including translation efficiency, protein stability, and degradation pathways, can lead to genuine differences between mRNA and protein abundance that may explain discordance between RT-PCR and antibody-based protein detection results . Epitope masking represents another common issue, where protein-protein interactions, post-translational modifications, or conformational changes may prevent antibody access to the recognition site in certain cellular contexts, leading to false-negative results in some assay formats but not others . Experimental comparison of native versus denaturing conditions can help identify such cases. Protein localization differences detected between antibody-based methods and fluorescent protein tagging approaches may reflect genuine biological phenomena, as tags can sometimes alter protein trafficking or localization; validation through subcellular fractionation followed by Western blotting provides an independent method to resolve such conflicts . For quantitative discrepancies, researchers should consider detection method sensitivities and dynamic ranges—mass spectrometry might detect low-abundance forms or modified versions of SPBC1289.13c that fall below antibody detection thresholds . When evaluating contradictory results, it is essential to assess the validation status of each method; newly developed techniques require more extensive validation than well-established protocols with documented specificity and sensitivity parameters .

What controls should be included when using SPBC1289.13c antibody in complex experimental designs?

Comprehensive controls are critical when designing experiments using SPBC1289.13c antibody, particularly for complex experimental setups investigating regulatory networks or protein interactions in fission yeast . A SPBC1289.13c deletion strain (if viable) or a strain with inducible depletion represents the gold-standard negative control for antibody specificity validation across all applications, demonstrating signal absence or reduction when the target protein is removed or depleted . For protein overexpression studies, a strain overexpressing SPBC1289.13c serves as a positive control, showing proportionally increased signal intensity in antibody detection methods . In co-immunoprecipitation experiments investigating protein interaction networks, isotype control antibodies (irrelevant rabbit IgG for this polyclonal rabbit-derived antibody) should be run in parallel to identify non-specific binding to the antibody class itself or to purification matrices . For chromatin immunoprecipitation experiments, input controls (pre-immunoprecipitation samples) and IgG controls are essential, while additional controls should include immunoprecipitation from cells where the target protein is tagged with an epitope recognized by a different well-characterized antibody for confirmation . When examining SPBC1289.13c involvement in stress responses or developmental transitions, time-course experiments should include appropriate time-matched controls to distinguish specific responses from general cellular changes . For quantitative applications, standard curves using recombinant SPBC1289.13c protein at known concentrations allow absolute quantification, while loading controls like tubulin or GAPDH in Western blots enable relative quantification across different samples .

How is research on SPBC1289.13c expected to evolve with advances in antibody technology?

Research on SPBC1289.13c is poised to benefit significantly from emerging advances in antibody technology that enhance specificity, sensitivity, and experimental versatility. Next-generation recombinant antibody development techniques may soon provide monoclonal alternatives to the current polyclonal SPBC1289.13c antibody, offering improved lot-to-lot consistency and potentially higher specificity for specific domains or conformational states of the protein . Single-domain antibodies (nanobodies) derived from camelid species represent a promising approach for creating smaller antibody fragments capable of accessing epitopes that might be sterically hindered for conventional antibodies, potentially revealing previously unstudied aspects of SPBC1289.13c function or interactions . Innovations in antibody engineering could produce bi-specific antibodies capable of simultaneously binding SPBC1289.13c and one of its interaction partners, enabling selective detection of specific protein complexes rather than total protein populations . Integration with proximity labeling techniques like BioID or APEX could transform how researchers map SPBC1289.13c interaction networks by identifying transient or weak interactions that are difficult to capture with traditional co-immunoprecipitation approaches . Additionally, antibodies conjugated to quantum dots or other photostable fluorophores will likely enhance live-cell and super-resolution imaging capabilities, providing unprecedented spatial resolution for studying SPBC1289.13c localization and dynamics in fission yeast cells . These technological developments, combined with increasing integration of computational approaches for antibody design and epitope prediction, promise to substantially expand the experimental toolkit available for investigating SPBC1289.13c function in fundamental cellular processes.

What are the potential applications of SPBC1289.13c research for understanding conserved eukaryotic processes?

Research on SPBC1289.13c using specific antibodies has significant potential for illuminating conserved eukaryotic processes due to fission yeast's position as a model organism with many core pathways shared with higher eukaryotes . If SPBC1289.13c functions within transcriptional regulatory networks, insights gained from studying its role in fission yeast could inform understanding of gene expression control mechanisms in more complex organisms, particularly if structural or functional homologs exist in mammals . The relatively simple genome of S. pombe, with approximately 5,100 genes compared to over 20,000 in humans, provides a less redundant system for identifying fundamental protein functions that may be obscured by functional redundancy in higher organisms . Detailed characterization of SPBC1289.13c protein interactions through antibody-based methods could reveal conserved protein complexes that have been maintained throughout eukaryotic evolution, pointing to essential cellular machinery deserving further study in human cells . If SPBC1289.13c is involved in cell cycle regulation, DNA repair, chromosome segregation, or cellular metabolism—processes highly conserved from yeast to humans—findings may have translational relevance for understanding human disease mechanisms . The antibody enables precise tracking of protein dynamics during cellular responses to environmental stresses, potentially uncovering evolutionarily conserved stress response pathways that could inform therapeutic strategies for conditions involving cellular stress in humans . Additionally, as a research tool for comparative biology studies, the SPBC1289.13c antibody could help establish which aspects of protein function and regulation have been conserved versus diverged throughout evolution, contributing to fundamental questions in evolutionary cell biology .

What is the recommended antibody validation framework for SPBC1289.13c studies?

A comprehensive validation framework for SPBC1289.13c antibody should begin with genetic validation using a SPBC1289.13c deletion strain (if viable) or knockdown strain, demonstrating antibody signal reduction or elimination that correlates with decreased protein expression . Orthogonal validation comparing antibody-based detection with an independent method, such as a differentially tagged version of SPBC1289.13c (e.g., GFP-tagged) detected with anti-GFP antibodies, provides confirmation across multiple detection systems . Independent antibody validation using a second SPBC1289.13c antibody raised against a different epitope should show concordant results in terms of protein detection pattern, subcellular localization, and experimental outcomes . Expression validation correlating antibody signal with known or experimentally manipulated expression levels (through inducible promoters or stress conditions reported to affect expression) demonstrates that the antibody signal accurately reflects protein abundance changes . Immunoblot analysis should show a predominant band of the expected molecular weight, with any additional bands characterized through mass spectrometry or other approaches to determine if they represent modified forms, cleavage products, or non-specific binding . Epitope mapping through peptide arrays or deletion constructs can precisely identify the antibody recognition site, valuable information for interpreting results when protein interactions or modifications might affect epitope accessibility . All validation data should be systematically documented with positive and negative controls included, and researchers should follow the recently established guidelines from the International Working Group for Antibody Validation to ensure their SPBC1289.13c antibody meets field-standard validation criteria .

What standardized reporting information should be included in publications using SPBC1289.13c antibody?

Publications utilizing SPBC1289.13c antibody should include comprehensive reporting information to ensure experimental reproducibility and transparent evaluation of results. Researchers must provide complete antibody identification details including manufacturer/source (e.g., Cusabio), catalog number (CSB-PA528479XA01SXV), lot number, antibody type (polyclonal), host species (rabbit), and immunogen information (recombinant Schizosaccharomyces pombe strain 972/ATCC 24843 SPBC1289.13c protein) . Detailed validation evidence specifically performed by the researchers should be described, including tests of specificity (Western blots showing band pattern, confirmation with knockout/knockdown strains) and sensitivity (detection limits determined through dilution series) . For each experimental application, precise methodology details must be reported: antibody dilutions used (e.g., 1:1000 for Western blot, 1:100 for immunofluorescence), incubation conditions (time, temperature, buffer composition), detection methods, and image acquisition parameters for microscopy or blot imaging . All experimental controls should be explicitly described, including negative controls (secondary antibody only, isotype controls, non-expressing samples) and positive controls (overexpression systems, purified proteins) . Any observed limitations of the antibody in specific applications should be candidly reported, such as fixation sensitivity, epitope masking under certain conditions, or cross-reactivity with closely related proteins . Additionally, for quantitative applications, researchers should include calibration methods and statistical approaches used for data analysis, along with representative images of raw data before any contrast enhancement or other post-processing .

The table below provides a standardized SPBC1289.13c antibody reporting checklist for researchers: