BUB3 Antibody

What is BUB3 and what is its functional significance in cell division?

BUB3 is a constitutively-expressed protein that functions as a mitotic checkpoint protein primarily in the nucleus. It serves as one of the core components of the spindle assembly checkpoint (SAC) alongside BUB1, preventing the onset of anaphase until all chromosomes have been correctly attached to microtubules during the cell cycle . The protein has a predicted molecular mass of approximately 37-38 kDa .

BUB3's significance in cell division cannot be overstated - cells lacking BUB3 exhibit an increased rate of chromosome mis-segregation, leading to aneuploidy, which further causes cell cycle delay or cell death . Studies have demonstrated that BUB3 null embryos treated with spindle-depolymerizing agents fail to arrest in metaphase and show increased mitotic disarray . This checkpoint mechanism is fundamentally important for maintaining genomic stability during cell division.

How is BUB3 localized throughout the cell cycle phases?

BUB3 demonstrates distinct localization patterns depending on the cell cycle phase. During interphase, BUB3 primarily localizes to the nucleus, unlike some other checkpoint proteins such as MPS1 which exhibits cytosolic localization . This nuclear localization is controlled by a specific nuclear localization signal (NLS) motif spanning amino acids 216-222 (Lys216 to Lys222) .

During the mitotic phase, BUB3 relocates to appear as discrete dots at the kinetochores, which can be visualized and confirmed by co-staining with centromeric markers like CREST antiserum or CENP-A . This dynamic localization pattern is crucial for BUB3's checkpoint function, as it needs to monitor kinetochore-microtubule attachments during mitosis. Immunofluorescence studies using GFP-tagged BUB3 have confirmed that this tagged version recapitulates the localization behavior of endogenous BUB3 in cells, appearing as discrete dots at kinetochores during prometaphase and prophase .

What protein interactions does BUB3 form during mitotic checkpoint signaling?

BUB3 forms critical interactions with multiple mitotic checkpoint proteins and kinetochore components that are essential for its checkpoint function. Research has demonstrated that BUB3 interacts with:

• MAD2 - A key checkpoint protein that helps prevent premature anaphase onset

• BubR1 - Another critical checkpoint protein involved in SAC signaling

• BUB1 - A kinase that functions alongside BUB3 in the core SAC mechanism

• KNL1 - A kinetochore protein that provides a platform for checkpoint protein assembly

• CENP-A - A centromeric histone variant that marks centromeres

These interactions are functionally significant and can be modulated by mutations in BUB3. For example, the QE mutation (where polar charged amino acid residues Lys266, Lys267, and Arg268 are mutated to polar uncharged Gln) impairs BUB3's interaction with BubR1, KNL1, and CENP-A, but not with MAD2 and BUB1 in HEK293T cells . Interestingly, these interaction patterns may vary between cell types, as the same mutation in HeLa cells impaired BUB3 interaction with CENP-A and KNL1 but not with MAD2, BUB1, and BubR1 .

What criteria should be considered when selecting a BUB3 antibody for research applications?

When selecting a BUB3 antibody for research applications, several critical criteria should be considered:

• Antibody specificity: The antibody should recognize BUB3 specifically without cross-reactivity to other proteins. Western blotting should show a single band at the expected molecular weight of approximately 37-38 kDa .

• Validated applications: Choose antibodies that have been validated for your specific applications (e.g., Western blotting, immunofluorescence, immunoprecipitation). For example, some BUB3 antibodies are specifically validated for immunofluorescence applications while others may be better suited for Western blotting .

• Species reactivity: Ensure the antibody recognizes BUB3 in your species of interest. Available antibodies show reactivity with human, mouse, and rat samples .

• Clone type: Consider whether a monoclonal (e.g., clone AB03/4E7-5) or polyclonal antibody better suits your research needs. Monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies may provide stronger signals by recognizing multiple epitopes .

• Immunogen information: Review the immunogen used to generate the antibody. Some BUB3 antibodies are raised against recombinant proteins covering specific amino acid regions, which may affect recognition of particular protein domains or mutants .

How can researchers validate the specificity of a BUB3 antibody?

Validating the specificity of a BUB3 antibody is crucial for ensuring reliable experimental results. Several approaches can be employed:

• RNA interference validation: Transfect cells with shRNAs targeting BUB3 and confirm decreased signal intensity by Western blotting. A specific antibody should show reduced band intensity proportional to the knockdown efficiency . Research has shown that when HeLa cells are transfected with shBUB3, a specific BUB3 antibody should demonstrate decreased band intensity at 37 kDa without affecting levels of other checkpoint proteins like BUB1, MAD2, and CENP-A .

• Recombinant protein expression: Express tagged BUB3 protein in cells and confirm that the antibody detects both endogenous and overexpressed protein at the expected sizes. GFP-tagged BUB3 constructs have been successfully used for this purpose .

• Immunoprecipitation followed by mass spectrometry: Immunoprecipitate proteins using the BUB3 antibody and confirm the presence of BUB3 by mass spectrometry.

• Cell cycle-dependent localization: Confirm that the antibody detects the expected nuclear localization during interphase and kinetochore localization during mitosis by immunofluorescence microscopy .

• Parallel testing with multiple antibodies: Use multiple antibodies targeting different epitopes of BUB3 and compare their staining patterns and Western blot results.

What is the significance of BUB3's nuclear localization signal for antibody selection and experimental design?

The nuclear localization signal (NLS) of BUB3, identified as the motif from Lys216 to Lys222, has significant implications for antibody selection and experimental design:

• Epitope masking concerns: Antibodies targeting epitopes within or near the NLS region might have reduced accessibility if this region is involved in protein-protein interactions or is masked by nuclear import machinery. This could lead to differences in antibody performance between fixed and live-cell applications .

• Mutant studies: When studying BUB3 mutants with deletions or modifications to the NLS (such as the Del216-222 mutant), researchers must ensure that their antibodies can still recognize the mutated protein. Antibodies targeting other regions of BUB3 would be preferable for these studies .

• Subcellular fractionation controls: For experiments involving nuclear/cytoplasmic fractionation, the NLS is a critical feature that determines BUB3 localization. Antibodies should be validated in fractionation experiments to confirm they can detect both nuclear and potentially cytoplasmic pools of BUB3 .

• Functional studies: Research has shown that deletion of the NLS (Del216-222) causes BUB3 to distribute in both the nucleus and cytoplasm, impairing its function in mitotic checkpoint signaling. Antibodies must be able to detect this altered localization when studying BUB3 function .

• Post-translational modifications: Consider whether post-translational modifications near the NLS might affect antibody recognition, especially if these modifications regulate nuclear import/export.

How can BUB3 antibodies be used to investigate mitotic checkpoint activation?

BUB3 antibodies can be powerful tools for investigating mitotic checkpoint activation through several advanced experimental approaches:

• Immunofluorescence-based mitotic indexing: BUB3 antibodies can be used alongside mitotic markers (e.g., phospho-histone H3) to quantify the proportion of cells arrested in mitosis following checkpoint activation. This approach can be used to compare checkpoint function between different cell lines or following experimental manipulations .

• Kinetochore recruitment dynamics: Using immunofluorescence with BUB3 antibodies, researchers can quantify the temporal recruitment of BUB3 to kinetochores during mitotic progression or following treatment with spindle poisons like nocodazole . The intensity of BUB3 at kinetochores correlates with checkpoint activation status.

• Co-immunoprecipitation studies: BUB3 antibodies can be used to immunoprecipitate BUB3 and its interacting partners under different conditions of checkpoint activation. This approach has revealed that mutations in BUB3 (such as the QE mutation) can alter interactions with proteins like BubR1, KNL1, and CENP-A during checkpoint signaling .

• Western blot analysis of checkpoint markers: Following mitotic arrest, BUB3 antibodies can be used alongside antibodies recognizing mitotic markers (cyclin B1, phospho-Ser 10 of histone H3) and phosphoepitopes (MPM2) to assess checkpoint activation biochemically .

• Live-cell imaging: When combined with GFP-tagged BUB3 constructs and complementary antibody staining of fixed cells, researchers can gain insights into the dynamic behavior of BUB3 during checkpoint activation and silencing .

What protocols are optimal for using BUB3 antibodies in co-immunoprecipitation experiments?

For optimal co-immunoprecipitation experiments with BUB3 antibodies, the following protocol elements are recommended based on published research:

Sample preparation:

• Treat cells with spindle poisons (e.g., nocodazole for 18 hours) to enrich for mitotic cells and maximize checkpoint protein interactions .

• Lyse cells in a buffer that preserves protein-protein interactions while efficiently extracting nuclear proteins (where BUB3 primarily localizes).

Immunoprecipitation procedure:

• For tagged BUB3 constructs: Use antibodies against the tag (e.g., GFP) for immunoprecipitation to avoid potential epitope masking issues .

• For endogenous BUB3: Use specific BUB3 antibodies that have been validated for immunoprecipitation applications.

• Include appropriate controls: IgG control, input sample, and potentially BUB3-depleted cells as a negative control .

Detection of interacting partners:

• Probe for known BUB3 interactors like BUB1, BubR1, MAD2, KNL1, and CENP-A .

• Use antibodies against these proteins that have been validated for Western blotting to ensure reliable detection.

Analysis considerations:

This protocol approach has successfully demonstrated differences in protein interaction patterns between wild-type BUB3 and mutants like QE in both HEK293T and HeLa cells .

What are the considerations for using BUB3 antibodies in cancer research applications?

When using BUB3 antibodies in cancer research applications, several important considerations should be taken into account:

• Prognostic marker evaluation: BUB3 has been suggested as a prognostic marker in prostate cancer, with cytoplasmic expression significantly correlated with recurrence . Antibodies must be validated specifically for immunohistochemistry applications on tissue sections with appropriate controls.

• Subcellular localization analysis: Since the cytoplasmic versus nuclear localization of BUB3 may have prognostic significance, antibodies must reliably detect BUB3 in both compartments . Validation should include known positive controls displaying both patterns.

• Expression level quantification: For studies comparing BUB3 expression levels between normal and cancer tissues, antibodies should demonstrate a linear detection range across relevant expression levels.

• Cell cycle considerations: Since BUB3 functions primarily during mitosis, interpretation of staining patterns should consider the proliferation status and cell cycle distribution of the analyzed tissue .

• Aneuploidy correlation studies: Given BUB3's role in preventing chromosome mis-segregation, antibodies can be used in conjunction with DNA content analysis to investigate correlations between BUB3 abnormalities and aneuploidy in cancer samples .

• Cross-species studies: For research involving animal models of cancer, antibodies with validated cross-reactivity to the relevant species should be selected .

What factors can affect BUB3 antibody staining patterns in immunofluorescence experiments?

Several factors can significantly influence BUB3 antibody staining patterns in immunofluorescence experiments:

• Cell cycle stage: BUB3 shows distinct localization patterns depending on the cell cycle phase - nuclear during interphase and at kinetochores during mitosis . Unsynchronized cell populations will show heterogeneous staining patterns reflecting these different localizations.

• Fixation method: Different fixation protocols can affect epitope accessibility. For BUB3, which has both nuclear and kinetochore localization, optimizing the fixation method is crucial. Paraformaldehyde fixation followed by permeabilization with detergents is commonly used, but methanol fixation might be preferable for certain antibodies .

• Antibody dilution: The recommended dilution range for different BUB3 antibodies can vary significantly. For example, some immunofluorescence applications recommend dilutions from 1:20-1:200 . Inadequate dilution can lead to high background, while excessive dilution may result in weak or undetectable signals.

• Antigen retrieval: For certain tissue sections or heavily fixed samples, antigen retrieval methods may be necessary to expose BUB3 epitopes.

• Co-staining considerations: When performing co-staining with other antibodies, potential cross-reactivity and appropriate controls should be considered. BUB3 is often co-stained with kinetochore markers like CENP-A or CREST antiserum for colocalization studies .

• Imaging parameters: For quantitative analysis of BUB3 staining intensity at kinetochores, consistent imaging parameters and proper background subtraction are essential.

How can inconsistent BUB3 detection in Western blotting be resolved?

When facing inconsistent BUB3 detection in Western blotting, researchers can implement several troubleshooting strategies:

• Sample preparation optimization:

  • Ensure complete nuclear protein extraction, as BUB3 is primarily nuclear during interphase .

  • Include phosphatase inhibitors in lysis buffers, as BUB3 function may be regulated by phosphorylation.

  • Use fresh samples when possible, as degradation can affect detection.

• Electrophoresis and transfer conditions:

  • Optimize gel percentage (10-12% is typically suitable for the 37-38 kDa BUB3 protein) .

  • Ensure complete transfer of proteins in the 35-40 kDa range.

  • Consider using PVDF membranes, which may offer better protein retention than nitrocellulose for some applications.

• Antibody selection and dilution:

  • For BUB3 Western blotting, validated antibodies show a single band at approximately 37-38 kDa .

  • Test different primary antibody dilutions, typically starting with 1:1000 as recommended for some BUB3 antibodies .

  • Increase incubation time (overnight at 4°C) for weaker antibodies.

• Enhanced detection methods:

  • For low abundance detection, consider using enhanced chemiluminescence (ECL) substrates with higher sensitivity.

  • Signal accumulation using longer exposure times may be required for weaker signals.

• Positive controls:

  • Include lysates from cells known to express BUB3 (e.g., HEK293 cells) .

  • Consider using recombinant BUB3 protein as a positive control.

What controls are essential when studying BUB3 mutants with antibody-based techniques?

When studying BUB3 mutants using antibody-based techniques, several essential controls must be included:

• Expression level verification:

  • Confirm that wild-type and mutant BUB3 proteins (e.g., Del216-222, QE mutant) are expressed at comparable levels to ensure fair comparison of their functional properties .

  • This verification should be performed by Western blotting using antibodies that recognize regions unaffected by the mutations.

• Antibody recognition validation:

  • For each BUB3 mutant, verify that the antibody still recognizes the mutated protein by Western blotting .

  • This is particularly important for deletion mutants or mutations near the antibody epitope.

• Cellular localization controls:

  • Include co-staining with nuclear markers (e.g., DAPI) and kinetochore markers (e.g., CENP-A) to assess proper localization .

  • For nuclear localization mutants like Del216-222, confirm altered localization patterns using immunofluorescence with appropriate nuclear/cytoplasmic markers .

• Functional rescue experiments:

  • When studying the function of BUB3 mutants, include experiments where endogenous BUB3 is depleted (e.g., using shRNA) and then rescued with shRNA-resistant wild-type or mutant BUB3 constructs .

  • This approach has been successfully used to demonstrate that the nuclear localization signal is essential for BUB3's function in mitotic checkpoint signaling .

• Protein interaction controls:

  • For co-immunoprecipitation experiments with BUB3 mutants, include wild-type BUB3 as a positive control and empty vector as a negative control .

  • Assess interactions with known partners like BUB1, BubR1, MAD2, KNL1, and CENP-A .

How do BUB3 antibodies contribute to understanding cancer progression mechanisms?

BUB3 antibodies are making significant contributions to understanding cancer progression mechanisms through several research approaches:

• Prognostic marker identification: Research has shown that cytoplasmic expression of BUB3 significantly correlates with cancer recurrence, potentially serving as a prognostic marker in prostate cancer . Antibody-based tissue staining allows researchers to evaluate this correlation across patient cohorts.

• Chromosomal instability mechanisms: Since cells lacking BUB3 have increased rates of chromosome mis-segregation leading to aneuploidy , antibodies enable researchers to investigate the relationship between BUB3 dysfunction and genomic instability in cancer. This includes quantitative assessments of BUB3 at kinetochores in cancer cell lines.

• Mitotic checkpoint impairment analysis: Cancer cells often exhibit checkpoint defects. BUB3 antibodies, used alongside mitotic markers, help researchers evaluate whether the spindle assembly checkpoint is compromised in specific cancer types through immunofluorescence and biochemical assays .

• Therapeutic response studies: By monitoring BUB3 expression and localization using specific antibodies, researchers can investigate how cancer cells respond to treatments targeting mitotic processes, such as taxanes or newer checkpoint inhibitors.

• Interaction with oncogenes and tumor suppressors: Co-immunoprecipitation experiments using BUB3 antibodies allow investigation of how BUB3 interacts with known oncogenes or tumor suppressors in different cancer contexts .

What emerging applications for BUB3 antibodies exist in developmental biology research?

Emerging applications for BUB3 antibodies in developmental biology research include:

• Embryonic cell division regulation: BUB3 is an essential component of spindle-assembly checkpoint signaling that operates during early embryogenesis . BUB3 antibodies allow researchers to track the activation and silencing of this checkpoint during critical developmental stages.

• Oocyte meiosis studies: BUB3 plays a role in promoting the establishment of correct kinetochore-microtubule (K-MT) attachments in mammalian oocyte meiosis . Antibodies enable visualization of BUB3 during these specialized cell divisions.

• Developmental aneuploidy investigation: Since BUB3 null embryos treated with spindle-depolymerizing agents fail to arrest in metaphase and show increased mitotic disarray , antibodies help researchers investigate the consequences of checkpoint dysfunction during development.

• Tissue-specific expression patterns: Immunohistochemistry with BUB3 antibodies allows mapping of expression patterns across different tissues during development, potentially revealing tissue-specific regulatory mechanisms.

• Stem cell division regulation: As stem cells must maintain genomic integrity through multiple divisions, BUB3 antibodies help researchers investigate whether checkpoint mechanisms operate differently in stem cell populations compared to differentiated cells.

• Model organism studies: With antibodies showing reactivity across species (human, mouse, rat) , comparative studies of BUB3 function across model organisms can reveal evolutionary conservation or divergence of checkpoint mechanisms during development.

How can BUB3 antibodies be used in conjunction with emerging technologies for mitotic research?

BUB3 antibodies can be powerfully combined with emerging technologies to advance mitotic research:

• Super-resolution microscopy: Conventional immunofluorescence studies have established BUB3's kinetochore localization , but super-resolution techniques (STORM, STED, SIM) with BUB3 antibodies can reveal precise substructural localization within the kinetochore, providing insights into checkpoint protein architecture.

• Live-cell imaging integration: By correlating live-cell imaging of fluorescently-tagged BUB3 with fixed-cell antibody staining, researchers can connect dynamic behaviors with molecular interactions during mitotic progression and checkpoint activation .

• Mass spectrometry-based proteomics: BUB3 antibodies can be used for immunoprecipitation coupled with mass spectrometry to discover novel interacting partners or post-translational modifications regulating BUB3 function in different cellular contexts .

• CRISPR/Cas9 genome editing validation: When creating BUB3 knockout or knock-in cell lines using CRISPR/Cas9, antibodies provide essential validation of successful editing through Western blotting and immunofluorescence.

• Single-cell analysis techniques: BUB3 antibodies compatible with flow cytometry or mass cytometry (CyTOF) enable researchers to correlate checkpoint protein levels with cell cycle stage or other parameters at the single-cell level.

• Proximity labeling approaches: By combining BUB3 antibodies with emerging proximity labeling techniques (BioID, APEX), researchers can map the local protein environment of BUB3 at different cell cycle stages or under different conditions of checkpoint activation.

These integrated approaches leverage the specificity of BUB3 antibodies alongside cutting-edge technologies to provide deeper insights into the molecular mechanisms of mitotic checkpoint regulation.