SPAC10F6.17c Antibody

What is SPAC10F6.17c and what is its functional significance?

SPAC10F6.17c is a protein found in Schizosaccharomyces pombe (strain 972/ATCC 24843), commonly known as fission yeast. This protein plays a significant role in regulating pyruvate dehydrogenase activity, which is central to cellular metabolism and energy production. Pyruvate dehydrogenase is a crucial enzyme complex that converts pyruvate to acetyl-CoA, linking glycolysis to the citric acid cycle. Understanding SPAC10F6.17c's regulatory function provides insights into yeast metabolic pathways and potentially broader eukaryotic metabolic regulation mechanisms. The protein is cataloged under UniProt number O14189 and Entrez Gene ID 2543002 .

What are the validated applications for SPAC10F6.17c Antibody?

SPAC10F6.17c Antibody has been validated for specific laboratory applications including Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) . These applications make it valuable for both quantitative detection and qualitative visualization of the target protein. For Western Blotting, the antibody enables detection of the target protein in complex mixtures following electrophoretic separation. In ELISA applications, it allows for quantification of SPAC10F6.17c in various sample types. The antibody's affinity purification ensures specificity for these applications, though researchers should validate performance in their specific experimental conditions.

What are the proper storage and handling recommendations for SPAC10F6.17c Antibody?

The SPAC10F6.17c Antibody should be stored at either -20°C or -80°C to maintain its integrity and functionality . For optimal performance, aliquoting the antibody upon receipt is recommended to minimize freeze-thaw cycles, which can degrade antibody quality. When handling the antibody, researchers should use proper sterile technique and avoid contamination. The antibody preparation includes 200μg antigens (for positive control), 1ml pre-immune serum (negative control), and affinity-purified rabbit polyclonal antibodies . These components should be stored separately according to the same temperature recommendations to preserve their functionality.

What controls should be included when using SPAC10F6.17c Antibody?

When conducting experiments with SPAC10F6.17c Antibody, appropriate controls are essential for result validation. The antibody is supplied with 200μg of antigens that serve as a positive control and 1ml of pre-immune serum that functions as a negative control . The positive control confirms antibody functionality, while the negative control helps identify non-specific binding. Additional recommended controls include: a loading control (e.g., housekeeping protein) for Western Blots; antibody-only controls without primary sample; and wild-type vs. SPAC10F6.17c knockout samples when available. Including these controls helps distinguish between specific signals and background, particularly important when investigating previously uncharacterized aspects of SPAC10F6.17c function.

How can SPAC10F6.17c Antibody be optimized for studying pyruvate dehydrogenase regulation?

Given that SPAC10F6.17c plays a role in regulating pyruvate dehydrogenase activity, this antibody can be instrumental in elucidating regulatory mechanisms. For optimal results, researchers should consider a multi-methodological approach combining immunoprecipitation with activity assays. Begin by establishing baseline pyruvate dehydrogenase activity in wild-type S. pombe using standard enzymatic assays. Then utilize the SPAC10F6.17c Antibody for immunoprecipitation to isolate protein complexes containing the target protein. This allows for identification of interaction partners through mass spectrometry. To study dynamic regulation, researchers can compare SPAC10F6.17c localization and associated protein complexes under various metabolic conditions (fermentation vs. respiration) using immunofluorescence combined with Western blotting. The antibody's affinity purification characteristics make it suitable for these complex applications when properly optimized.

What methodologies are recommended for analyzing SPAC10F6.17c expression under stress conditions?

Investigating SPAC10F6.17c expression patterns under various stress conditions requires carefully designed experimental protocols. Begin with exposure of S. pombe cultures to different stressors (oxidative stress, nutrient limitation, temperature shifts) at multiple time points. For protein level analysis, prepare cell lysates following standard protocols optimized for yeast cells, ensuring complete protease inhibition. Use the SPAC10F6.17c Antibody in Western blot analysis with 20-50μg of total protein per lane, adjusting primary antibody concentration between 1:1000-1:5000 for optimal signal-to-noise ratio. For transcriptional analysis, complement protein studies with RT-qPCR targeting SPAC10F6.17c mRNA. This dual approach allows correlation between transcriptional and translational responses to stress. Immunofluorescence microscopy using the antibody can further reveal stress-induced changes in subcellular localization, providing insights into functional adaptations.

What are the considerations for cross-reactivity when using SPAC10F6.17c Antibody in comparative studies?

While the SPAC10F6.17c Antibody is raised against a recombinant Schizosaccharomyces pombe protein, researchers conducting comparative studies across species must carefully validate cross-reactivity . Begin with in silico analysis to identify potential homologs in target species using tools like BLAST and assess sequence conservation in the immunogenic regions. Prior to large-scale experiments, conduct preliminary Western blots with lysates from multiple species alongside positive controls from S. pombe. If cross-reactivity is observed, validate specificity through competitive binding assays using recombinant proteins. For closely related yeast species, consider the evolutionary conservation of pyruvate dehydrogenase regulation pathways when interpreting results. If cross-reactivity is insufficient, alternative approaches such as epitope tagging of homologous proteins may be necessary for comparative studies.

How can SPAC10F6.17c Antibody be integrated into ChIP protocols to study potential DNA interactions?

If investigation suggests SPAC10F6.17c may interact with DNA or chromatin-associated complexes (directly or indirectly), the antibody can be adapted for Chromatin Immunoprecipitation (ChIP) applications. Begin with crosslinking optimization using 1% formaldehyde for 10-15 minutes at room temperature, followed by quenching with glycine. After cell lysis using glass beads optimized for yeast, sonicate chromatin to fragments of 200-500bp. For immunoprecipitation, use 2-5μg of SPAC10F6.17c Antibody per reaction, incubating overnight at 4°C with rotation. The antibody's rabbit polyclonal nature provides multiple epitope recognition, potentially increasing ChIP efficiency. Following standard washing procedures, reverse crosslinks and purify DNA for analysis by qPCR or sequencing. Include appropriate controls: input chromatin, IgG control precipitation, and positive control targets if available. This methodology allows mapping of potential SPAC10F6.17c associations with specific genomic regions.

What are the key technical specifications of commercially available SPAC10F6.17c Antibody?

The following table summarizes the essential specifications of SPAC10F6.17c Antibody based on available commercial information:

This antibody has been developed with specificity toward the SPAC10F6.17c protein from S. pombe, with its production involving immunization of rabbits with a recombinant form of the target protein .

How can researchers troubleshoot non-specific binding with SPAC10F6.17c Antibody?

Non-specific binding is a common challenge when working with polyclonal antibodies. If experiencing high background or unexpected bands with SPAC10F6.17c Antibody, implement the following methodological approaches. First, optimize blocking conditions by testing different blocking agents (5% BSA, 5% non-fat milk, or commercial blocking reagents) and extending blocking time to 2 hours at room temperature. Second, adjust antibody concentration by performing a dilution series (1:500 to 1:5000) to identify the optimal working concentration. Third, increase washing stringency by adding 0.1-0.3% Tween-20 to wash buffers and extending wash durations. For persistent non-specific binding, pre-adsorption can be effective: incubate the diluted antibody with the provided pre-immune serum at a 1:10 ratio for 2 hours before application. Finally, if available, validate specificity using SPAC10F6.17c knockout samples or competitive blocking with recombinant protein. These methodological adjustments should significantly reduce non-specific binding while preserving specific signal detection.

What are the recommended protocols for using SPAC10F6.17c Antibody in co-immunoprecipitation studies?

For co-immunoprecipitation (Co-IP) studies investigating SPAC10F6.17c protein interactions, the following protocol is recommended. Harvest S. pombe cells in mid-log phase and lyse in a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail. Clear lysate by centrifugation at 14,000g for 10 minutes at 4°C. Pre-clear 500μg-1mg of total protein with Protein A/G beads for 1 hour at 4°C. Incubate pre-cleared lysate with 2-5μg of SPAC10F6.17c Antibody overnight at 4°C with gentle rotation. Add 30-50μl of Protein A/G beads and incubate for an additional 2-4 hours. Wash precipitates 4-5 times with lysis buffer containing reduced detergent concentrations (0.1% NP-40). Elute bound proteins by boiling in SDS sample buffer. Analyze by SDS-PAGE followed by Western blotting with antibodies against suspected interaction partners. Always include a negative control using pre-immune serum or non-specific IgG, and validate interactions through reciprocal Co-IP when possible.

How can SPAC10F6.17c Antibody contribute to understanding yeast metabolic regulation pathways?

Given SPAC10F6.17c's role in regulating pyruvate dehydrogenase activity, the antibody presents valuable opportunities for investigating metabolic regulation in yeast. Researchers can implement a multi-faceted experimental approach beginning with immunoprecipitation using SPAC10F6.17c Antibody followed by mass spectrometry to identify protein interaction networks. This approach can reveal how SPAC10F6.17c connects to broader metabolic signaling pathways. Western blot analysis using the antibody can track SPAC10F6.17c expression and post-translational modifications under different carbon sources (glucose, glycerol, ethanol) to understand its regulatory dynamics. Combining these protein-level studies with metabolomics analysis allows correlation between SPAC10F6.17c activity and metabolite profiles. Additionally, researchers can employ the antibody in immunofluorescence studies to track subcellular localization changes in response to metabolic shifts, providing spatial context to its regulatory function. This comprehensive approach can establish SPAC10F6.17c's position within yeast metabolic regulatory networks.

What methodological considerations are important when using SPAC10F6.17c Antibody in comparative evolutionary studies?

When employing SPAC10F6.17c Antibody for evolutionary studies comparing pyruvate dehydrogenase regulation across fungal species, several methodological considerations are essential. First, sequence alignment analysis should be performed to identify conserved regions in SPAC10F6.17c homologs across fungal species, focusing on epitope regions recognized by the antibody. For experimental validation, prepare standardized protein extracts from multiple species using identical extraction protocols to ensure comparable results. Western blotting should be performed with gradient gels (4-12%) to accommodate potential size variations in homologs, with the SPAC10F6.17c Antibody used at 1:1000 dilution initially, adjusting as needed for each species. Include S. pombe extracts as positive controls in all experiments. For species showing cross-reactivity, immunoprecipitation followed by mass spectrometry can confirm target identity. Complement antibody-based studies with functional assays measuring pyruvate dehydrogenase activity across species to correlate protein detection with enzymatic function. This integrated approach provides insights into the evolutionary conservation of SPAC10F6.17c's regulatory mechanisms.

How can researchers integrate SPAC10F6.17c Antibody data with transcriptomics and proteomics datasets?

Integrating SPAC10F6.17c Antibody-generated data with broader -omics approaches requires systematic methodology. Begin with parallel sample preparation where cultures for antibody-based studies (Western blot, immunoprecipitation) are grown alongside cultures for RNA-seq and global proteomics under identical conditions. For data integration, normalize Western blot quantification of SPAC10F6.17c against multiple housekeeping proteins and correlate with mRNA expression levels from transcriptomics. Immunoprecipitation using SPAC10F6.17c Antibody followed by mass spectrometry identifies interacting partners, which can be mapped onto protein-protein interaction networks constructed from global proteomics data. This creates a SPAC10F6.17c-centered interactome. For advanced analysis, employ machine learning algorithms to identify patterns correlating SPAC10F6.17c levels/modifications with global expression changes across experimental conditions. Visualization tools like Cytoscape can create integrated network maps highlighting SPAC10F6.17c's position within metabolic and regulatory pathways. This comprehensive approach contextualizes antibody-derived data within the broader cellular system, providing deeper insights into SPAC10F6.17c function.