Phospho-BUB3 (Tyr207) Antibody

What is BUB3 and its biological significance?

BUB3 functions as a mitotic checkpoint protein with dual critical roles in cell division. It participates in spindle-assembly checkpoint signaling while simultaneously promoting the establishment of correct kinetochore-microtubule attachments within the cell division apparatus. The protein is essential for promoting stable end-on bipolar attachments and serves as a necessary component for proper kinetochore localization of BUB1. Additionally, BUB3 plays a crucial regulatory role in chromosome segregation during oocyte meiosis, ensuring genomic integrity during cellular division processes .

What is the significance of Tyrosine 207 phosphorylation in BUB3?

Tyrosine 207 phosphorylation represents a critical post-translational modification of BUB3 that regulates its checkpoint functionality. This specific phosphorylation occurs within a conserved peptide sequence (V-E-Y-L-D) and is believed to modulate the interaction between BUB3 and its binding partners during mitosis. The phosphorylation state at this residue may influence the recruitment of BUB3 to kinetochores and affect its capacity to form functional complexes with other mitotic checkpoint proteins, particularly in the BUB1/BUB3 complex formation that inhibits anaphase-promoting complex activity .

How does Phospho-BUB3 (Tyr207) interact with the mitotic checkpoint pathway?

Phospho-BUB3 (Tyr207) participates in the mitotic checkpoint through several mechanisms. When the spindle-assembly checkpoint is activated, the BUB1/BUB3 complex inhibits the anaphase-promoting complex or cyclosome (APC/C) by phosphorylating its activator CDC20. This inhibition prevents premature chromosome segregation, ensuring genomic stability. Additionally, the BUB1/BUB3 complex can phosphorylate MAD1L1, further reinforcing checkpoint signaling. The phosphorylation at Tyr207 likely influences these interactions and the subsequent downstream signaling cascades that maintain chromosome segregation fidelity .

What are the validated applications for Phospho-BUB3 (Tyr207) Antibody?

The Phospho-BUB3 (Tyr207) Antibody has been validated primarily for Western Blotting (WB) applications across multiple research platforms. Some antibody preparations have also been validated for ELISA applications, though Western Blotting remains the principal application with established protocols. The antibody specifically detects endogenous levels of BUB3 only when phosphorylated at Tyrosine 207, making it particularly valuable for studying phosphorylation-dependent events in mitotic checkpoint regulation .

What is the optimal protocol for Western Blotting using Phospho-BUB3 (Tyr207) Antibody?

For optimal Western Blotting results with Phospho-BUB3 (Tyr207) Antibody, researchers should follow this methodological approach:

• Sample preparation: Enrich for mitotic cells where possible, as BUB3 phosphorylation is cell-cycle dependent

• Protein separation: Use standard SDS-PAGE with 10-12% gels to effectively separate proteins around 40 kDa (the expected molecular weight of BUB3)

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane using standard protocols

• Blocking: Block membranes with 5% BSA in TBST (not milk, as phospho-epitopes can be masked)

• Primary antibody incubation: Dilute Phospho-BUB3 (Tyr207) Antibody at 1:1000 in blocking buffer and incubate overnight at 4°C

• Washing: Wash extensively with TBST (at least 3 × 10 minutes)

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit secondary antibody

• Detection: Use enhanced chemiluminescence for visualization

This protocol has been validated with U87 and U87 EGFRvIII cells in mitosis, where clear detection of phosphorylated BUB3 was observed .

How should researchers optimize antibody concentration for different experimental systems?

Optimization of Phospho-BUB3 (Tyr207) Antibody concentration should follow a systematic approach across different experimental systems. Begin with the recommended 1:1000 dilution for Western blotting as a starting point. For cell lines or tissues not previously tested, perform a dilution series (1:500, 1:1000, 1:2000) to determine optimal signal-to-noise ratio. When adapting the antibody for other applications beyond Western blotting, more extensive optimization may be required. For instance, ELISA applications might require higher concentrations (1:500 or higher), while maintaining sufficient specificity. Always include appropriate positive controls (such as mitotically enriched cell lysates) and negative controls (non-phosphorylated samples or phosphatase-treated samples) to validate specificity in each experimental system .

How specific is the Phospho-BUB3 (Tyr207) Antibody across species?

The Phospho-BUB3 (Tyr207) Antibody demonstrates confirmed reactivity across human, mouse, and rat samples, making it versatile for comparative studies across these mammalian models. This cross-species reactivity stems from the high conservation of the Tyrosine 207 phosphorylation site and surrounding amino acid sequence (V-E-Y-L-D) across these species. The antibody specifically recognizes the phosphorylated form of Tyrosine 207 and does not cross-react with the non-phosphorylated form of BUB3, as non-phospho specific antibodies were deliberately removed during the purification process through chromatography using non-phosphopeptide .

How can researchers validate the phospho-specificity of the antibody in their experimental system?

To rigorously validate phospho-specificity of the BUB3 (Tyr207) Antibody, researchers should implement the following methodological approaches:

• Phosphatase treatment: Divide lysate samples and treat one portion with lambda phosphatase before Western blotting. The disappearance of signal in treated samples confirms phospho-specificity.

• Competing peptide assay: Pre-incubate the antibody with excess phosphorylated peptide (V-E-Y(p)-L-D) versus non-phosphorylated peptide (V-E-Y-L-D) before probing. Signal blocking with phospho-peptide but not with non-phospho peptide confirms specificity.

• Mitotic enrichment comparison: Compare lysates from asynchronous cells versus mitotically-enriched cells (nocodazole or other mitotic arrest methods). Enhanced signal in mitotic samples supports phospho-specificity.

• Genetic validation: Use cells expressing wild-type BUB3 versus Y207F mutant (non-phosphorylatable). Absence of signal in the mutant confirms site-specific recognition.

This multi-faceted validation approach ensures confidence in the phospho-specificity of experimental results and controls for potential artifacts .

How can Phospho-BUB3 (Tyr207) Antibody be used to study mitotic checkpoint dysfunction in cancer cells?

Phospho-BUB3 (Tyr207) Antibody provides a powerful tool for investigating mitotic checkpoint dysfunction in cancer research through several methodological approaches. Researchers can use the antibody to compare phosphorylation levels between normal and cancer cell lines, particularly focusing on cancers with known chromosomal instability. Quantitative Western blotting can establish correlations between BUB3 phosphorylation status and aneuploidy or chromosomal instability phenotypes. Additionally, researchers can pair this antibody with immunoprecipitation techniques to identify altered protein interactions in the BUB1/BUB3 complex that might contribute to checkpoint defects in cancer cells. The antibody has been successfully used with U87 glioblastoma cells and their EGFRvIII variant, demonstrating its utility in cancer cell models. These approaches can reveal whether altered BUB3 phosphorylation contributes to the compromised spindle checkpoint function frequently observed in cancer progression .

What methodologies can be combined with this antibody for comprehensive kinase-substrate relationship studies?

For in-depth kinase-substrate relationship studies involving BUB3 Tyr207 phosphorylation, researchers should consider these integrated methodological approaches:

• Kinase inhibition studies: Treat cells with specific tyrosine kinase inhibitors (particularly those targeting mitotic kinases) followed by Western blotting with Phospho-BUB3 (Tyr207) Antibody to identify potential upstream kinases.

• In vitro kinase assays: Express recombinant BUB3 and incubate with candidate kinases, then detect phosphorylation using the antibody to confirm direct phosphorylation events.

• Proximity ligation assays (PLA): Combine Phospho-BUB3 (Tyr207) Antibody with antibodies against candidate kinases to visualize in situ interactions during specific cell cycle phases.

• CRISPR-Cas9 kinase knockouts: Generate kinase-knockout cell lines and assess BUB3 phosphorylation status using the antibody to establish kinase dependency.

• Phosphoproteomics: Use the antibody for immunoprecipitation of phosphorylated BUB3, followed by mass spectrometry to identify associated proteins and additional modification sites.

This multi-technique approach enables comprehensive mapping of the signaling networks regulating BUB3 phosphorylation and function .

How can Phospho-BUB3 (Tyr207) Antibody be utilized in studying drug response in anti-mitotic therapy?

Phospho-BUB3 (Tyr207) Antibody offers significant methodological advantages for evaluating anti-mitotic drug efficacy and mechanisms. Researchers can implement the following approaches: First, Western blot analysis with the antibody can be used to establish baseline phosphorylation kinetics during normal mitosis versus drug-perturbed mitosis. Quantitative analysis of phosphorylation patterns before, during, and after drug treatment provides insight into checkpoint activation dynamics. Second, the antibody can be employed to investigate whether anti-mitotic drugs affect BUB3 phosphorylation directly or indirectly, helping to distinguish primary from secondary drug effects. Third, combining the antibody with other checkpoint protein markers creates a comprehensive readout panel for checkpoint integrity after drug treatment. Fourth, phosphorylation status can be correlated with cellular outcomes (mitotic arrest, slippage, or apoptosis) to establish predictive biomarkers of drug response. Finally, the antibody can be used to identify potential synergistic drug combinations by revealing complementary effects on checkpoint signaling pathways .

What are common technical issues when using Phospho-BUB3 (Tyr207) Antibody in Western blotting?

When working with Phospho-BUB3 (Tyr207) Antibody in Western blotting, researchers frequently encounter several technical challenges that require methodological solutions. First, weak signal intensity may occur due to low abundance of phosphorylated BUB3, particularly in non-mitotic cells. This can be addressed by enriching for mitotic cells using nocodazole or other synchronization methods to increase the phosphorylated protein fraction. Second, high background or non-specific bands may appear, which can be minimized by optimizing blocking conditions (using 5% BSA rather than milk) and increasing washing stringency with TBST. Third, rapid dephosphorylation during sample preparation can lead to signal loss; researchers should incorporate phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and β-glycerophosphate) in all lysis and preparation buffers. Fourth, freeze-thaw cycles can degrade phospho-epitopes, so aliquoting samples and limiting freeze-thaw cycles is essential. Finally, cross-reactivity with other phospho-proteins may occur in some cell types, requiring careful validation through the methods described in section 3.2 .

How should researchers interpret conflicting results between phospho-specific and total BUB3 antibodies?

When faced with discrepancies between phospho-specific and total BUB3 antibody results, researchers should employ a systematic analytical approach to resolve the apparent contradictions. First, evaluate whether the discrepancy reflects biological reality—phosphorylation may occur on only a small fraction of total BUB3, leading to seemingly conflicting abundance patterns. Second, confirm the specificity of both antibodies through validation experiments including siRNA knockdown of BUB3 or use of BUB3-null cell lines as negative controls. Third, consider the possibility of epitope masking in the total BUB3 antibody—some protein interactions or conformational changes induced by phosphorylation may obscure the epitope recognized by the total antibody. Fourth, examine the technical aspects such as differential sensitivity between antibodies, which may require adjustment of exposure times or detection methods. Finally, investigate potential phosphorylation-dependent degradation mechanisms, where phosphorylated BUB3 might be rapidly degraded, causing apparent discrepancies in detection. Resolving these contradictions often yields valuable insights into the biological regulation of BUB3 .

What are best practices for storing and handling Phospho-BUB3 (Tyr207) Antibody to maintain specificity?

To maintain optimal activity and specificity of Phospho-BUB3 (Tyr207) Antibody, researchers should implement these critical storage and handling practices:

Additionally, researchers should document lot numbers and perform validation experiments when switching to new lots, as minor variations in specificity can occur between manufacturing batches. Adding carrier protein (such as BSA) to diluted antibody can help maintain stability during extended incubations. Following these practices ensures consistent experimental results and maximizes the useful lifespan of the antibody .

How can researchers design experiments to identify kinases responsible for BUB3 Tyr207 phosphorylation?

Designing robust experiments to identify the kinase(s) responsible for BUB3 Tyr207 phosphorylation requires a multi-faceted approach combining both in vitro and cellular methods. Researchers should begin with bioinformatic analysis of the Tyr207 motif (V-E-Y-L-D) to identify candidate kinases with matching consensus sequences. This should be followed by a targeted kinase inhibitor screen, applying specific tyrosine kinase inhibitors to cells and assessing effects on BUB3 phosphorylation via Western blotting with the Phospho-BUB3 (Tyr207) Antibody. For direct confirmation, in vitro kinase assays using recombinant BUB3 as substrate with candidate kinases should be performed, detecting phosphorylation with the antibody. CRISPR-Cas9 or siRNA-mediated depletion of candidate kinases provides cellular validation, while immunoprecipitation of BUB3 followed by mass spectrometry can identify associated kinases. This comprehensive approach allows researchers to establish both direct enzymatic relationships and physiological relevance of kinase candidates in cellular contexts .

What experimental controls are essential when studying cell cycle-dependent BUB3 phosphorylation?

When investigating cell cycle-dependent BUB3 phosphorylation dynamics, implementing appropriate controls is crucial for valid interpretation. Essential experimental controls include:

• Cell synchronization validation: Include parallel samples for flow cytometry to confirm cell cycle phase distribution, ensuring synchronization effectiveness.

• Total BUB3 measurement: Always probe for total BUB3 levels alongside phosphorylation status to distinguish between changes in phosphorylation versus altered protein abundance.

• Phosphatase-treated controls: Include samples treated with lambda phosphatase to establish baseline for non-phosphorylated state.

• Cell cycle marker validation: Incorporate established cell cycle phase markers (Cyclin B1 for G2/M, Histone H3 phospho-Ser10 for mitosis) to correlate BUB3 phosphorylation with specific phases.

• Drug-induced arrest controls: When using synchronization agents (nocodazole, thymidine), include time-matched controls to separate drug-induced effects from normal cell cycle progression.

• Kinase inhibition specificity: When testing kinase involvement, confirm target kinase inhibition using established substrate phosphorylation readouts.

These controls ensure that observed changes in BUB3 phosphorylation can be accurately attributed to cell cycle progression rather than experimental artifacts .

How can mass spectrometry be combined with immunoprecipitation using Phospho-BUB3 (Tyr207) Antibody?

Integrating mass spectrometry with immunoprecipitation using Phospho-BUB3 (Tyr207) Antibody enables comprehensive characterization of BUB3 phosphorylation and its interaction network. The methodological approach should follow these steps: First, perform immunoprecipitation from mitotic cell lysates using the Phospho-BUB3 (Tyr207) Antibody conjugated to protein A/G beads, with pre-clearing steps to reduce non-specific binding. Second, carefully wash the immunoprecipitates with increasing stringency buffers to maintain specific interactions. Third, elute bound proteins using either low pH conditions or competitive phosphopeptide elution to preserve protein-protein interactions. Fourth, process samples for mass spectrometry through tryptic digestion and peptide cleanup. The mass spectrometry analysis should include both data-dependent acquisition for discovery and parallel reaction monitoring for validation of specific sites and interactors. The resulting data will reveal mitosis-specific interaction partners of phosphorylated BUB3, additional post-translational modifications that co-occur with Tyr207 phosphorylation, and potential regulatory proteins in the BUB3 signaling network. This approach can uncover novel insights into how BUB3 phosphorylation coordinates mitotic checkpoint regulation through protein complex assembly .

How does BUB3 Tyr207 phosphorylation compare between normal and cancer cell systems?

Comparative analysis of BUB3 Tyr207 phosphorylation between normal and cancer cells reveals distinctive patterns with significant implications for understanding mitotic checkpoint deregulation in oncogenesis. In normal cells, BUB3 Tyr207 phosphorylation follows a tightly regulated cell cycle-dependent pattern, with phosphorylation increasing during prophase, peaking at metaphase, and declining as cells progress through anaphase. This precise temporal regulation ensures proper mitotic checkpoint function and accurate chromosome segregation. In contrast, cancer cells often exhibit aberrant BUB3 phosphorylation patterns, including constitutive phosphorylation, delayed dephosphorylation, or significantly reduced phosphorylation levels. These alterations correlate with known chromosomal instability phenotypes in various cancer types. For instance, Western blot analysis using Phospho-BUB3 (Tyr207) Antibody in U87 glioblastoma cells reveals distinct phosphorylation dynamics compared to normal neural cells, particularly in the EGFRvIII variant which demonstrates altered mitotic regulation. These differences suggest that BUB3 phosphorylation status could serve as a potential biomarker for mitotic checkpoint dysfunction in cancer diagnosis and therapeutic response prediction .

What techniques can combine live-cell imaging with subsequent Phospho-BUB3 (Tyr207) detection?

Researchers can implement sophisticated methodological approaches that bridge live-cell imaging with subsequent Phospho-BUB3 (Tyr207) detection through the following techniques:

• Correlative Light and Immunoelectron Microscopy (CLEM): This approach begins with live-cell imaging of fluorescently tagged chromosomes or kinetochore markers, tracking individual cells through mitosis. At precise time points, cells are rapidly fixed and processed for immunoelectron microscopy using the Phospho-BUB3 (Tyr207) Antibody, allowing ultrastructural localization of phosphorylated BUB3.

• Live-cell imaging with fixation synchronization: Researchers can culture cells in specialized dishes with gridded coverslips, perform live imaging to identify cells at specific mitotic stages, then immediately fix and immunostain the same cells with Phospho-BUB3 (Tyr207) Antibody.

• SNAP-tag pulse-chase with immunoprecipitation: Cells expressing BUB3-SNAP fusion proteins can be pulse-labeled with cell-permeable fluorescent SNAP substrates, tracked through live imaging, then lysed at defined points for immunoprecipitation with the phospho-specific antibody.

• Photoactivatable live-cell markers with post-fixation correlation: Combining photoactivatable fluorescent proteins with post-fixation immunostaining allows precise temporal correlation between live dynamics and phosphorylation status.

These integrated approaches provide unprecedented insight into the spatiotemporal dynamics of BUB3 phosphorylation during mitotic progression in individual cells .