pof15 Antibody

What is pof15 Antibody and what organism is it specific for?

pof15 Antibody (product code CSB-PA884635XA01SXV) is a polyclonal antibody raised in rabbits against recombinant Schizosaccharomyces pombe (strain 972 / ATCC 24843, commonly known as fission yeast) pof15 protein. This antibody specifically recognizes the pof15 protein (UniProt No. Q9HDX6) and has been validated for species reactivity with S. pombe strain 972 . The antibody has been developed through antigen affinity purification techniques and is provided in liquid form with appropriate preservatives for research applications.

What are the validated applications for pof15 Antibody?

pof15 Antibody has been validated for specific research applications including enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB). These applications enable researchers to detect and quantify pof15 protein expression levels in fission yeast samples . For Western blotting applications, the antibody can be used to identify the target protein after separation by gel electrophoresis, while ELISA applications allow for quantitative analysis of pof15 in solution-based samples.

What are the optimal storage conditions for maintaining pof15 Antibody activity?

To maintain optimal activity of pof15 Antibody, storage at either -20°C or -80°C is recommended immediately upon receipt. It is critical to avoid repeated freeze-thaw cycles as these can significantly decrease antibody activity and lead to protein degradation . The antibody is supplied in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative, which helps maintain stability during proper storage conditions.

How might pof15 interact with the F-BAR protein Cdc15 in S. pombe cell division pathways?

Based on current research into S. pombe cellular mechanisms, the relationship between pof15 and the F-BAR protein Cdc15 represents an important area for investigation. Cdc15 plays an essential role in cytokinesis by attaching the cytokinetic ring (CR) to the plasma membrane (PM) . The function of Cdc15 is regulated through phosphorylation by multiple cell polarity kinases, including Pom1, Kin1, Pck1, and Shk1 .

Considering pof15's presence in the same organism, researchers should investigate potential interactions between pof15 and the Cdc15 phosphorylation pathway, possibly as a regulatory factor or participant in the phase separation process. Experimental approaches might include co-immunoprecipitation studies using the pof15 Antibody to determine whether pof15 directly interacts with Cdc15 or influences its phosphorylation state during different cell cycle phases.

What technical considerations should be addressed when using pof15 Antibody in phase separation studies of S. pombe proteins?

Recent research has shown that dephosphorylated Cdc15 undergoes liquid-liquid phase separation to form droplets in vitro that can recruit binding partners . When designing experiments to investigate whether pof15 might participate in similar phase separation processes or influence Cdc15 condensation, several technical considerations should be addressed:

• Buffer composition must be carefully controlled, as phase separation is highly sensitive to ionic strength, pH, and temperature

• Antibody concentration should be optimized to avoid artificial aggregation while maintaining detection sensitivity

• Distinguishing between specific binding and non-specific interactions requires appropriate controls

A methodological approach would involve using pof15 Antibody in immunofluorescence studies to determine if pof15 co-localizes with Cdc15 condensates in vivo, particularly during cell division. Additionally, in vitro reconstitution experiments could determine if pof15 affects the phase separation properties of Cdc15.

How can phosphorylation state analysis of potential pof15 interactions be optimized using this antibody?

Given that multiple kinases regulate Cdc15 phosphorylation with distinct but overlapping cohorts of phosphorylation sites , researchers investigating pof15's relationship to phosphorylation-dependent processes should consider:

• Combining pof15 Antibody immunoprecipitation with phosphoproteomic analysis to identify phosphorylation sites on interacting proteins

• Using phospho-specific antibodies in conjunction with pof15 Antibody for co-localization studies

• Employing phosphatase treatments in control experiments to distinguish phosphorylation-dependent interactions

The methodological approach should include western blotting with phospho-specific antibodies after pof15 Antibody immunoprecipitation, and mass spectrometry analysis to identify phosphorylation sites on potential pof15-interacting proteins. This approach would help elucidate whether pof15 preferentially interacts with phosphorylated or dephosphorylated forms of target proteins like Cdc15.

What is the optimal protocol for using pof15 Antibody in Western blotting applications with S. pombe lysates?

When using pof15 Antibody for Western blotting applications with S. pombe lysates, researchers should follow this optimized protocol:

• Sample preparation:

  • Harvest S. pombe cells during the appropriate growth phase

  • Lyse cells using glass bead disruption in a buffer containing protease inhibitors (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA, protease inhibitor cocktail)

  • Clear lysate by centrifugation at 15,000 × g for 15 minutes at 4°C

• Gel electrophoresis and transfer:

  • Separate proteins on a 10-12% SDS-PAGE gel

  • Transfer to PVDF membrane using standard protocols (25 mM Tris, 192 mM glycine, 20% methanol)

• Antibody incubation:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with pof15 Antibody at an optimized dilution (starting at 1:1000) overnight at 4°C

  • Wash 3 times with TBST, 5 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody

  • Develop using enhanced chemiluminescence

• Controls and validation:

  • Include negative control (non-expressing strain)

  • Consider using tagged pof15 expression constructs as positive controls

  • Validate specificity with peptide competition assays

This methodological approach ensures specific detection of pof15 while minimizing background and non-specific signals.

How can researchers troubleshoot non-specific binding when using pof15 Antibody in immunoprecipitation studies?

When encountering non-specific binding issues in immunoprecipitation studies with pof15 Antibody, researchers should implement the following troubleshooting strategies:

• Optimize antibody concentration:

  • Titrate antibody amounts from 1-5 μg per reaction

  • Use protein A/G beads at 20-50 μl bed volume per reaction

• Modify washing conditions:

  • Increase salt concentration in wash buffers (150 mM to 300 mM NaCl)

  • Add mild detergents (0.1% Triton X-100)

  • Perform additional wash steps (5-6 washes instead of 3)

• Pre-clear lysates:

  • Incubate lysates with protein A/G beads without antibody for 1 hour at 4°C

  • Remove beads by centrifugation before adding pof15 Antibody

• Use appropriate controls:

  • Include IgG control from the same species (rabbit)

  • Perform parallel IPs with lysates from pof15 deletion strains

By systematically applying these approaches, researchers can significantly reduce non-specific binding and increase the signal-to-noise ratio in pof15 immunoprecipitation experiments.

What experimental design is recommended for investigating potential interactions between pof15 and phosphorylation-regulated proteins like Cdc15?

To effectively investigate potential interactions between pof15 and phosphorylation-regulated proteins like Cdc15, researchers should implement a multi-faceted experimental design:

• Co-immunoprecipitation studies:

  • Perform reciprocal co-IPs using both pof15 Antibody and Cdc15 antibodies

  • Compare interactions under various phosphorylation conditions (±kinase inhibitors, ±phosphatase treatment)

  • Analyze precipitates by western blotting and mass spectrometry

• Genetic interaction analysis:

  • Create pof15 deletion or overexpression strains

  • Cross with strains containing Cdc15 phosphomutants

  • Analyze phenotypes related to cytokinesis and cell division

• Microscopy studies:

  • Use pof15 Antibody for immunofluorescence in fixed cells

  • Compare localization with Cdc15-GFP fusion proteins

  • Examine co-localization during different cell cycle stages

• In vitro reconstitution:

  • Express and purify recombinant pof15 and Cdc15

  • Perform binding assays under controlled phosphorylation conditions

  • Analyze phase separation behavior in the presence of both proteins

This comprehensive approach allows researchers to determine whether and how pof15 may interact with phosphorylation-regulated proteins like Cdc15, potentially revealing novel regulatory mechanisms in S. pombe cell division.

What statistical approaches are recommended for analyzing pof15 expression levels across different experimental conditions?

When analyzing pof15 expression levels detected by the antibody across different experimental conditions, researchers should consider these statistical approaches:

A methodological approach would involve:

• Testing data for normality using Shapiro-Wilk or Kolmogorov-Smirnov tests

• Applying appropriate transformations if needed (log, square root)

• Performing the appropriate statistical test based on experimental design

• Calculating effect sizes in addition to p-values

• Establishing biological significance thresholds for pof15 expression changes

Researchers should report both statistical significance and biological relevance when interpreting changes in pof15 expression levels across conditions.

How should researchers interpret potential cross-reactivity of pof15 Antibody with other F-box proteins in S. pombe?

When interpreting potential cross-reactivity of pof15 Antibody with other F-box proteins in S. pombe, researchers should implement the following analytical framework:

• Sequence homology analysis:

  • Perform bioinformatic analysis of sequence similarity between pof15 and other S. pombe F-box proteins

  • Identify regions of high homology that might result in cross-reactivity

• Validation experiments:

  • Test antibody reactivity in pof15 deletion strains

  • Perform peptide competition assays with recombinant pof15 and similar F-box proteins

  • Use epitope mapping to identify the specific binding region

• Data interpretation guidelines:

  • Consider bands at unexpected molecular weights as potential cross-reactive proteins

  • Correlate western blot results with mRNA expression data from RT-qPCR

  • Implement additional antibodies targeting different epitopes for confirmation

This methodological approach ensures accurate interpretation of results and proper identification of true pof15 signals versus potential cross-reactivity with other F-box family members in experimental systems.

What are promising approaches for investigating pof15's role in relation to phase separation phenomena in S. pombe?

Building on recent findings that dephosphorylated Cdc15 undergoes liquid-liquid phase separation , researchers interested in investigating pof15's potential role in phase separation phenomena should consider these approaches:

• In vitro phase separation assays:

  • Express and purify recombinant pof15

  • Test its intrinsic phase separation properties under various conditions

  • Examine how pof15 affects phase separation of known condensate-forming proteins like Cdc15

• Live-cell imaging techniques:

  • Create fluorescently tagged pof15 strains

  • Use high-resolution microscopy to track potential condensate formation

  • Apply techniques like FRAP (Fluorescence Recovery After Photobleaching) to measure dynamics

• Proteomics of isolated condensates:

  • Use pof15 Antibody to immunoprecipitate potential condensates

  • Identify components by mass spectrometry

  • Compare composition under different cellular conditions