SPCP25A2.03 Antibody

What is SPCP25A2.03 antibody and what organism does it target?

SPCP25A2.03 antibody is a polyclonal antibody specifically designed to detect the SPCP25A2.03 protein in Schizosaccharomyces pombe (strain 972 / ATCC 24843), commonly known as fission yeast . The antibody is produced in rabbits using a recombinant SPCP25A2.03 protein as the immunogen, which increases its specificity for the target protein . According to the available data, this antibody corresponds to UniProt accession number Q9URT2, which represents the specific protein sequence it recognizes . The antibody has been purified using antigen affinity methods, which enhances its specificity by reducing cross-reactivity with unrelated proteins . As with all research antibodies, validation of specificity in the context of specific experimental conditions is essential before proceeding with critical experiments .

What are the physical properties and storage conditions for SPCP25A2.03 antibody?

SPCP25A2.03 antibody is supplied in liquid form with specific buffer components designed to maintain stability and activity during storage and use . The storage buffer contains 50% glycerol and 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative to prevent microbial contamination during storage . For optimal longevity and performance, the antibody should be stored at either -20°C or -80°C, and repeated freeze-thaw cycles should be avoided as they can compromise antibody integrity and function . When handling the antibody, it's advisable to aliquot the stock solution into smaller volumes upon first thaw to minimize freeze-thaw cycles that could lead to protein denaturation and loss of activity . The antibody is provided as unconjugated IgG, which offers flexibility for different detection methods based on secondary antibody selection .

What applications has SPCP25A2.03 antibody been validated for?

According to the product information, SPCP25A2.03 antibody has been validated for specific research applications including Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) . These validation studies ensure that the antibody performs reliably in these specific experimental contexts when used according to recommended protocols . For Western blotting applications, the antibody can be used to detect the target protein from yeast cell lysates, providing information about protein expression levels and molecular weight . When using this antibody for the first time in a particular experimental system, researchers should conduct preliminary validation experiments including appropriate positive and negative controls as recommended by antibody validation guidelines . Similar to other research antibodies, optimization of experimental conditions such as antibody dilution, incubation time, and blocking reagents may be necessary to achieve optimal results .

What controls should be included when validating SPCP25A2.03 antibody for research use?

When validating SPCP25A2.03 antibody for research applications, multiple controls should be incorporated to ensure reliable results and proper interpretation of data . Primary validation should include a positive control consisting of samples known to express the target protein (Schizosaccharomyces pombe extracts) and negative controls such as samples from organisms or cell lines that do not express the target (e.g., mammalian cells like COS7) . A critical negative control is the use of non-immune IgG from the same species (rabbit) at the same concentration as the primary antibody to identify potential non-specific binding of the secondary detection system . As demonstrated in published antibody validation studies, comparing the banding pattern between positive samples, negative samples, and isotype controls helps distinguish specific signals from background or cross-reactivity . Additionally, if possible, genetic controls such as knockout or knockdown samples provide the most stringent validation of antibody specificity, though this may be challenging for essential genes in S. pombe .

What are the recommended protocols for using SPCP25A2.03 antibody in Western blotting?

For Western blotting applications using SPCP25A2.03 antibody, several protocol optimizations may enhance detection specificity and sensitivity . Sample preparation should involve thorough lysis of S. pombe cells using appropriate buffers containing protease inhibitors to prevent protein degradation during extraction . Based on standard Western blotting protocols, proteins should be separated by SDS-PAGE and transferred to an appropriate membrane (PVDF or nitrocellulose) using standard transfer conditions . For blocking, 5% non-fat dry milk in TBST or similar blocking solutions can be used to reduce non-specific binding, similar to protocols validated for other antibodies . The primary antibody concentration should be optimized through titration experiments, typically starting with dilutions in the range of 1:500 to 1:2000, with overnight incubation at 4°C . After washing steps with TBST buffer, an appropriate HRP-conjugated anti-rabbit secondary antibody should be applied, followed by detection using enhanced chemiluminescence (ECL) or similar detection systems .

How can SPCP25A2.03 antibody be validated for specificity in experimental systems?

Validating the specificity of SPCP25A2.03 antibody requires a multi-faceted approach similar to established antibody validation methodologies . One essential validation approach involves testing the antibody against samples with known expression patterns, comparing wild-type S. pombe extracts with strains lacking the target protein if available . Peptide competition assays, where the antibody is pre-incubated with excess immunizing peptide before application to the sample, can confirm binding specificity by demonstrating signal reduction when the antibody's binding sites are blocked . Western blot analysis should reveal a band of the expected molecular weight corresponding to the target protein in positive samples, with minimal or no cross-reactivity bands in negative control lanes . For research applications beyond Western blotting, such as immunofluorescence or immunoprecipitation, additional validation steps specific to each technique should be performed, following similar principles to those used for validating other antibodies in these applications . Researchers should document all validation steps thoroughly as recommended by initiatives promoting research antibody validation standards .

How can SPCP25A2.03 antibody be used in studying protein-protein interactions?

SPCP25A2.03 antibody can be employed in various techniques to investigate protein-protein interactions involving the target protein in S. pombe . Immunoprecipitation (IP) using this antibody can pull down the target protein along with its interacting partners, which can then be identified through mass spectrometry or Western blot analysis with antibodies against suspected binding partners . Co-immunoprecipitation (Co-IP) experiments can be designed where cell lysates are incubated with the SPCP25A2.03 antibody to precipitate protein complexes, followed by SDS-PAGE separation and immunoblotting with antibodies against candidate interacting proteins . For more sophisticated analyses, techniques like proximity ligation assays (PLA) could potentially be adapted using this antibody in combination with antibodies against putative interacting partners, though this would require additional validation . When designing such experiments, appropriate controls are essential, including negative controls using non-immune rabbit IgG and validation of IP efficiency using a portion of the precipitate for detection with the same antibody .

What are best practices for optimizing immunofluorescence experiments with SPCP25A2.03 antibody?

While the product information specifically validates SPCP25A2.03 antibody for ELISA and Western blotting applications, researchers may explore its utility in immunofluorescence studies following established optimization protocols . For immunofluorescence applications in S. pombe, cells should be fixed using methods that preserve antigen structure while allowing antibody access, such as formaldehyde fixation followed by cell wall digestion with enzymes like zymolyase . Permeabilization conditions should be optimized to allow antibody penetration while maintaining cellular structures, typically using detergents like Triton X-100 or methanol . Blocking solutions containing BSA or normal serum can reduce non-specific binding, and antibody dilutions should be empirically determined starting with manufacturer recommendations or 1:100-1:500 ranges . Similar to the approach demonstrated for SNAP25 antibody validation, comparing staining patterns between positive samples (S. pombe) and negative control samples lacking the target protein provides critical validation of specificity in immunofluorescence applications .

How can SPCP25A2.03 antibody be applied in studying post-translational modifications?

SPCP25A2.03 antibody may be utilized to investigate potential post-translational modifications (PTMs) of the target protein through several experimental approaches . For detecting phosphorylation states, researchers could perform immunoprecipitation with SPCP25A2.03 antibody followed by Western blotting with phospho-specific antibodies (such as anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibodies) similar to approaches used with PKC motif antibodies . Alternatively, two-dimensional gel electrophoresis followed by Western blotting with SPCP25A2.03 antibody can reveal different isoforms or modified versions of the target protein based on charge and molecular weight shifts . For studying ubiquitination, immunoprecipitation with SPCP25A2.03 antibody followed by immunoblotting with anti-ubiquitin antibodies could reveal if the target protein undergoes this modification, similar to approaches documented for other proteins . These experiments should include appropriate controls such as samples treated with phosphatase inhibitors (for phosphorylation studies) or proteasome inhibitors (for ubiquitination studies) to preserve the modified forms of the protein .

How can SPCP25A2.03 antibody be used in comparative studies across yeast species?

The SPCP25A2.03 antibody is generated against a specific protein from Schizosaccharomyces pombe, but researchers may explore its cross-reactivity with orthologous proteins in related yeast species through careful experimental design . Cross-reactivity testing should begin with sequence alignment analysis to identify potential orthologs in other yeast species with high sequence homology to the S. pombe SPCP25A2.03 protein . Western blot analysis using protein extracts from various yeast species (such as S. cerevisiae or other Schizosaccharomyces species) can determine if the antibody recognizes related proteins across species boundaries . When conducting such cross-reactivity studies, it is essential to include positive controls (S. pombe extracts) alongside the test samples to confirm proper antibody function and to establish relative binding affinities . If cross-reactivity is observed, further validation through techniques such as immunoprecipitation followed by mass spectrometry can confirm the identity of the recognized proteins in different species .

What approaches can be used to combine SPCP25A2.03 antibody with other molecular tools for comprehensive protein studies?

Integrating SPCP25A2.03 antibody with complementary molecular biology techniques can provide comprehensive insights into the target protein's function, localization, and regulation . Combining immunoprecipitation using this antibody with mass spectrometry analysis can identify not only interacting proteins but also post-translational modifications and protein variants, similar to approaches used in studying other protein complexes . Researchers could design experiments that combine this antibody with CRISPR-Cas9 gene editing of the target gene to validate antibody specificity and study protein function under various genetic modifications . Multiplexed immunofluorescence approaches, similar to those described for coronavirus antibodies, could potentially be developed using SPCP25A2.03 antibody alongside antibodies against other cellular markers to study colocalization patterns . For temporal studies of protein expression or modification, the antibody could be used in combination with techniques such as fluorescence recovery after photobleaching (FRAP) or photoactivatable fluorescent protein tagging to track dynamic protein behaviors .

How can SPCP25A2.03 antibody contribute to understanding conserved protein functions across species?

SPCP25A2.03 antibody can serve as a valuable tool in comparative evolutionary studies examining conserved protein functions across different species through carefully designed experimental approaches . Researchers can use this antibody to immunoprecipitate the target protein from S. pombe and identify interacting partners, then compare these interaction networks with known protein complexes in other model organisms to identify functionally conserved modules . Complementation studies, where the human ortholog (if identified) is expressed in S. pombe mutants lacking the endogenous protein, followed by immunoprecipitation with the antibody, can reveal functional conservation across evolutionary distances . When analyzing conserved post-translational modifications, the antibody can be used to immunoprecipitate the protein from S. pombe under various conditions, with subsequent mass spectrometry analysis to identify modification sites that can be compared to known regulatory sites in orthologs from other species . These evolutionary approaches require thorough bioinformatic analysis to identify true orthologs and paralogs across species, complemented by experimental validation using this antibody in combination with other molecular biology techniques .

What are common troubleshooting steps for Western blot experiments using SPCP25A2.03 antibody?

When encountering issues with Western blot experiments using SPCP25A2.03 antibody, several systematic troubleshooting approaches can help identify and resolve common problems . For weak or absent signals, researchers should verify protein extraction efficiency, increase antibody concentration, extend incubation times, or use more sensitive detection reagents, similar to optimization approaches described for other antibodies . High background issues can be addressed by optimizing blocking conditions (trying different blocking agents like BSA instead of milk), increasing washing stringency, or diluting the antibody further, following established Western blot troubleshooting protocols . Multiple or unexpected bands might indicate protein degradation (address by adding more protease inhibitors during extraction), post-translational modifications (verify with specific inhibitors), or potential cross-reactivity (validate with additional controls) . For batch-to-batch variation issues, researchers should maintain consistent experimental conditions, include positive control samples with known reactivity in each experiment, and consider preparing larger antibody aliquots from a single lot for long-term studies .

How should SPCP25A2.03 antibody performance be monitored over time?

Maintaining consistent antibody performance throughout a research project requires regular quality control measures similar to established protocols for antibody validation . Researchers should prepare a standard positive control sample (e.g., S. pombe extract with known expression of the target protein) in sufficient quantity to use across multiple experiments as a reference for antibody performance . When a new lot of antibody is received, side-by-side comparison with the previous lot using identical samples and protocols should be performed to document any sensitivity or specificity differences . Regular testing of antibody activity is recommended, particularly for aliquots stored for extended periods, by performing standard Western blot analysis with consistent sample loading and imaging parameters to detect any reduction in signal strength over time . Maintaining detailed records of antibody performance, including images of Western blots with standardized exposure settings, provides valuable reference data to identify potential degradation or variation in antibody function .

What are recommended practices for validating antibody specificity for SPCP25A2.03 across different experimental applications?

Comprehensive validation of SPCP25A2.03 antibody across different experimental applications requires a systematic approach integrating multiple strategies as demonstrated in antibody validation studies . For application-specific validation, researchers should first confirm antibody performance in the manufacturer-validated applications (ELISA and Western blot) before extending to other techniques such as immunoprecipitation or immunofluorescence . When adapting the antibody to new applications, validation should include genetic controls (when available) such as comparing wild-type S. pombe with strains where the target gene has been deleted or significantly downregulated . Orthogonal validation approaches, where the antibody results are compared with data from antibody-independent methods such as fluorescent protein tagging or RNA expression analysis, provide additional confidence in antibody specificity . For comprehensive validation across multiple applications, researchers should document the specific conditions optimized for each technique, including sample preparation methods, antibody dilutions, incubation conditions, and detection systems, following the guidelines established for rigorous antibody validation .