BUB3.2 Antibody

What is the optimal application for BUB3 antibody detection in cell cycle studies?

BUB3 antibodies are particularly effective for studying spindle assembly checkpoint components during mitosis. For optimal results, immunofluorescence (IF/ICC) applications at 1:50-1:200 dilutions provide excellent visualization of BUB3 localization at kinetochores during prometaphase . Western blotting (WB) at 1:500-1:2000 dilutions can effectively quantify BUB3 protein levels across different cell cycle stages . For investigating BUB3's dynamic localization during mitosis, live-cell imaging techniques paired with fluorescently-tagged BUB3 antibodies allow for temporal analysis of checkpoint activation and resolution .

How can I validate BUB3 antibody specificity in my experimental system?

Validating BUB3 antibody specificity requires multiple complementary approaches. First, perform Western blot analysis using cell lysates from multiple cell lines (such as HeLa, Jurkat, and 293T cells) to confirm detection of the expected 37-38 kDa band . Second, implement siRNA knockdown or CRISPR-Cas9 knockout of BUB3 as negative controls to verify signal specificity . Third, compare staining patterns with multiple BUB3 antibodies from different clones and manufacturers to confirm consistent localization patterns. Finally, recombinant expression of tagged BUB3 protein can serve as a positive control to confirm antibody recognition of the target protein .

What are the recommended cell lines for studying BUB3 functions using antibody-based techniques?

For studying BUB3 functions, several validated cell lines have demonstrated reliable antibody reactivity and biological relevance. HeLa cells show strong endogenous BUB3 expression and distinct kinetochore localization during mitosis, making them ideal for immunofluorescence studies . Jurkat cells provide consistent Western blot results with clear BUB3 detection at approximately 37-38 kDa . MCF7 and HL-60 cell lines have been validated as positive samples for BUB3 detection and are suitable for studying checkpoint activation in cancer contexts . For mouse studies, spleen, ovary, brain, and lung tissues have shown reliable BUB3 antibody reactivity and can be used for comparative analyses of BUB3 expression and function across different tissue types .

How can BUB3 antibodies be effectively used to study BUB3-protein interactions in checkpoint signaling?

To study BUB3's interactions with other checkpoint proteins, co-immunoprecipitation (co-IP) experiments using BUB3 antibodies can effectively capture protein complexes. Research shows that BUB3 interacts with key checkpoint proteins including Mad2, Mad3, and Cdc20 . For optimal co-IP results, cell lysates should be prepared from mitotically arrested cells using nocodazole treatment (typically 100 ng/ml for 16-18 hours) . Alternatively, for studying interactions in S-phase, hydroxyurea arrest protocols can be implemented as demonstrated in previous studies .

The choice of lysis buffer is critical - a buffer containing 50 mM Tris pH 7.6, 150 mM NaCl, 0.1% Triton X-100, and protease inhibitors preserves most BUB3 protein complexes . When analyzing results, it's important to note that some interactions (like BUB3-Cdc20) are dependent on other checkpoint proteins such as Mad1 and Mad2, requiring careful experimental design and appropriate controls . For detecting weaker or transient interactions, crosslinking approaches using formaldehyde (0.1-1%) prior to cell lysis can stabilize complexes for more comprehensive detection .

What are the critical factors for detecting BUB3 at kinetochores using immunofluorescence techniques?

Successful detection of BUB3 at kinetochores requires attention to several critical factors. The timing of fixation is crucial - BUB3 shows strongest kinetochore localization during prometaphase, so synchronizing cells or enriching for mitotic populations using nocodazole (100 ng/ml) or other mitotic inhibitors improves detection . The fixation method significantly impacts epitope accessibility; paraformaldehyde fixation (4% for 10-15 minutes) followed by permeabilization with 0.1% Triton X-100 preserves BUB3 localization while maintaining kinetochore structure .

For co-localization studies, combining BUB3 antibodies with kinetochore markers (like CENP-A or CREST serum) helps confirm the specific localization pattern . The antibody dilution must be carefully optimized - typically 1:50-1:200 for immunofluorescence applications . Finally, image acquisition parameters are critical; using high-resolution confocal microscopy with z-stack acquisition allows precise localization analysis of BUB3 at kinetochore structures during different mitotic stages .

How can I investigate the functional relationship between BUB3 and other checkpoint proteins using antibody-based approaches?

Investigating functional relationships between BUB3 and other checkpoint proteins requires combining antibody detection with functional perturbation experiments. For studying BUB3's relationship with BUB1, use co-immunostaining to analyze their co-localization patterns at kinetochores, noting that BUB3 is necessary for BUB1 kinetochore localization . Sequential immunoprecipitation experiments can reveal the composition of different BUB3-containing complexes, distinguishing between BUB3-BUB1-MAD1 and BUB3-CDC20-MAD2 interactions .

For functional studies, combine siRNA knockdown of one checkpoint component (e.g., MAD1 or MAD2) with immunofluorescence or Western blot analysis of BUB3 to assess dependency relationships . The data indicates that MAD1 and MAD2 are required for robust BUB3-CDC20 interactions, with MAD2 being indispensable for BUB3 association with both CDC20 and MAD1 . To investigate the role of BUB3 in the inhibition of APC/C, combine BUB3 antibody detection with ubiquitination assays or APC/C activity measurements in cellular extracts under various checkpoint conditions .

What controls should be included when using BUB3 antibodies for Western blotting?

Rigorous control experiments are essential for reliable Western blot results with BUB3 antibodies. Positive controls should include lysates from cells with confirmed BUB3 expression, such as HeLa, Jurkat, or 293T cells, where BUB3 appears as a band at approximately 37-38 kDa . Loading controls with housekeeping proteins (β-actin or GAPDH) are crucial for normalizing BUB3 expression levels between samples .

Negative controls should include lysates from BUB3-depleted cells (using siRNA or CRISPR-Cas9), which should show significant reduction in signal intensity . For antibody specificity validation, include primary antibody omission and isotype controls (using matched IgG from the same species) . When analyzing BUB3 expression changes across experimental conditions, include a dynamic range control with a dilution series of a reference sample to ensure signal linearity . Finally, when using HRP-labeled secondary antibodies, standardize the detection protocol with consistent exposure times to facilitate accurate quantitative comparisons across multiple experiments .

How can I optimize fixation and permeabilization protocols for BUB3 immunofluorescence studies?

Optimization of fixation and permeabilization protocols is critical for successful BUB3 immunofluorescence studies, as improper procedures can compromise epitope recognition. For adherent cell lines like HeLa, a sequential approach works best: fix cells with 4% paraformaldehyde in PBS for 10 minutes at room temperature, followed by three 5-minute washes with PBS containing 0.1% Triton X-100 (PBST) . Post-fixation blocking with 5% non-fat dried milk in PBST for 30 minutes reduces background signal .

Different epitopes may require adjusted protocols - for detecting BUB3 at kinetochores, methanol fixation (-20°C for 10 minutes) sometimes provides better results by exposing certain epitopes . When co-staining for multiple proteins, test different fixation methods on separate samples first, as some antibody combinations may have incompatible fixation requirements. For suspension cells, cytospin preparation followed by the same fixation protocol ensures proper cell morphology preservation . Regardless of the protocol, maintaining consistent fixation timing is essential, as overfixation can mask epitopes while underfixation may compromise cellular structure .

What are the most reliable quantification methods for analyzing BUB3 protein levels in Western blot experiments?

For reliable quantification of BUB3 protein levels, several methodological considerations must be addressed. Densitometric analysis should be performed using specialized software (ImageJ, ImageLab, etc.) on non-saturated images with appropriate background subtraction . Normalization to loading controls (β-actin, GAPDH) is essential for accurate comparison between samples, preferably using the ratio of BUB3 to loading control signal intensities .

Standard curves generated from serial dilutions of control lysates help establish the linear range of detection and validate quantification accuracy . For comparing BUB3 levels across multiple experimental conditions, include an internal reference sample on each blot to normalize between experiments . When detecting subtle changes in expression levels, chemiluminescence detection with multiple exposure times ensures capturing signals within the linear range . Finally, statistical analysis should include multiple biological replicates (minimum n=3) with appropriate statistical tests to determine significance of observed changes in BUB3 protein levels .

How can BUB3 antibodies be utilized to investigate mitotic checkpoint defects in cancer cells?

BUB3 antibodies serve as powerful tools for investigating mitotic checkpoint abnormalities in cancer. Immunohistochemistry using optimized BUB3 antibodies (1:200-1:500 dilution) on cancer tissue microarrays can reveal aberrant expression patterns across different cancer types . Research indicates that cytoplasmic expression of BUB3 correlates significantly with cancer recurrence, particularly in prostate cancer, making subcellular localization analysis a critical application . For mechanistic studies, combine BUB3 immunofluorescence with cell cycle markers to quantify the percentage of cells with proper kinetochore localization of BUB3 in cancer versus normal cells .

Western blot analysis of BUB3 protein levels across cancer cell lines can identify correlations between BUB3 expression and chromosomal instability phenotypes . For functional studies, pair BUB3 antibody detection with live-cell imaging of chromosome segregation to correlate BUB3 localization defects with mitotic errors in cancer cells . Advanced applications include chromatin immunoprecipitation (ChIP) using BUB3 antibodies to investigate potential non-canonical functions of BUB3 in gene regulation, which may contribute to cancer progression through mechanisms beyond its established role in chromosome segregation .

What approaches can be used to study post-translational modifications of BUB3 using specific antibodies?

Studying post-translational modifications (PTMs) of BUB3 requires specialized antibody-based approaches. When investigating phosphorylation events, phospho-specific BUB3 antibodies can be developed against known or predicted phosphorylation sites, though these must be rigorously validated using phosphatase treatments of control samples . Alternatively, immunoprecipitate BUB3 using general BUB3 antibodies followed by Western blotting with pan-phospho-antibodies (anti-phospho-serine/threonine/tyrosine) to detect modification status .

For detecting ubiquitination, immunoprecipitate BUB3 under denaturing conditions (1% SDS, 95°C) to disrupt protein-protein interactions, followed by Western blotting with anti-ubiquitin antibodies . Mass spectrometry analysis of immunoprecipitated BUB3 provides comprehensive PTM mapping, identifying modification sites and their relative abundance across different cellular conditions . For temporal analysis of BUB3 modifications during cell cycle progression, synchronize cells at different cell cycle stages (using thymidine, nocodazole, or other synchronization agents) followed by BUB3 immunoprecipitation and PTM analysis .

How can BUB3 antibodies be applied in high-throughput screening applications for mitotic regulators?

BUB3 antibodies can be effectively implemented in high-throughput screening approaches to identify novel mitotic regulators and potential therapeutic targets. Automated immunofluorescence microscopy platforms using BUB3 antibodies (1:100-1:200 dilution) can screen chemical or genetic libraries for compounds or genes that alter BUB3 kinetochore localization or protein levels . For higher throughput, in-cell Western assays in 96 or 384-well formats using infrared-labeled secondary antibodies provide quantitative measurement of BUB3 levels across large sample sets .

Flow cytometry-based approaches combining BUB3 antibody staining with DNA content analysis can rapidly identify conditions that affect the mitotic checkpoint, screening thousands of conditions in single experiments . For functional screening, combine BUB3 antibody detection with phenotypic readouts such as micronuclei formation or aneuploidy markers to identify compounds that disrupt proper chromosome segregation . Data analysis requires sophisticated image analysis algorithms that can extract multiple parameters from BUB3 staining patterns, including intensity, subcellular localization, and co-localization with other checkpoint proteins .

What are the key differences between monoclonal and polyclonal BUB3 antibodies for specific research applications?

The choice between monoclonal and polyclonal BUB3 antibodies significantly impacts experimental outcomes. Monoclonal antibodies like the rabbit recombinant monoclonal EPR5319(2) (ab133699) or mouse monoclonal AB03/4E7-5 offer superior specificity for a single epitope, ensuring consistent lot-to-lot reproducibility ideal for long-term studies . These antibodies excel in applications requiring high specificity, such as distinguishing BUB3 from related proteins or when background must be minimized . Conversely, polyclonal antibodies like the rabbit polyclonal CAB6536 recognize multiple epitopes on BUB3, providing stronger signals through epitope multiplicity and greater tolerance to protein denaturation, making them advantageous for detecting native proteins in immunoprecipitation experiments .

For Western blotting applications, monoclonal antibodies typically provide cleaner results with less background, though they may be more sensitive to epitope masking from protein modifications . For immunofluorescence applications, polyclonal antibodies often provide stronger signals but may show more batch-to-batch variation, while monoclonals ensure consistent staining patterns across experiments . When studying homologous proteins across species, polyclonal antibodies may offer broader cross-reactivity, while monoclonals provide more species-specific detection . The experimental design should dictate antibody selection, with critical experiments validated using both antibody types when possible.

How do different host species affect BUB3 antibody performance in various applications?

The host species used for BUB3 antibody production significantly influences performance across applications. Rabbit-derived BUB3 antibodies, such as the rabbit recombinant monoclonal (ab133699) and rabbit polyclonal (CAB6536), typically demonstrate high affinity and specificity, making them excellent for detecting low-abundance BUB3 in complex samples . Mouse-derived antibodies like clone AB03/4E7-5 provide advantages in multi-color immunofluorescence experiments where co-staining with other rabbit antibodies is required .

Host species selection becomes particularly important when working with tissue samples - using antibodies raised in species distinct from the experimental tissue prevents endogenous antibody cross-reactivity . For example, when studying mouse tissues, rabbit or goat-derived BUB3 antibodies are preferable to avoid detection of endogenous mouse immunoglobulins by anti-mouse secondary antibodies . Different host species may recognize distinct epitopes on BUB3, providing complementary information when used in parallel . Additionally, the isotype of the antibody (IgG, IgM, etc.) impacts protocol optimization - IgG2b antibodies like clone AB03/4E7-5 typically require different secondary antibody dilutions than IgG antibodies for optimal signal-to-noise ratios .

What methodological considerations are important when using BUB3 antibodies for comparative studies across species?

When conducting comparative studies of BUB3 across species, several methodological considerations ensure valid cross-species comparisons. First, evaluate sequence homology between species - human BUB3 shares high sequence conservation with mouse and rat homologs, enabling cross-reactivity for many antibodies . The immunogen sequence is critical - antibodies raised against full-length recombinant human BUB3 (amino acids 1-328) typically show broader cross-reactivity than those targeting smaller, potentially species-specific epitopes .

Validation experiments must be performed individually for each species using positive controls from relevant tissues (e.g., mouse spleen, ovary, brain, and lung have demonstrated BUB3 expression) . Optimization of detection protocols for each species is essential - different dilutions may be required for optimal results across species, with typical ranges of 1:500-1:2000 for Western blotting and 1:50-1:200 for immunofluorescence . For proteins with species-specific post-translational modifications, select antibodies recognizing conserved, unmodified regions to ensure consistent detection . Finally, when quantitatively comparing BUB3 levels between species, always include species-specific positive controls on the same blot or slide to normalize for potential differences in antibody affinity across species .