BUB1B Antibody

What are the primary applications for BUB1B antibodies in research?

BUB1B antibodies are widely used in multiple applications including:

These applications enable researchers to detect BUB1B expression patterns in various experimental systems, with particularly strong validation in cell lines such as HeLa, K-562, SKOV-3, A-549, MCF7, and DU145 .

How should BUB1B antibodies be optimized for immunohistochemistry staining?

For optimal IHC results with BUB1B antibodies:

• Use antigen retrieval with TE buffer pH 9.0 as the primary method

• Alternatively, citrate buffer pH 6.0 can be used if results are suboptimal

• Begin with a dilution range of 1:250-1:1000 and titrate for your specific tissue

• Human cervical and colon cancer tissues serve as reliable positive controls

• Include negative controls (isotype controls or tissues known to lack BUB1B expression)

Recent studies have successfully used these protocols to demonstrate that BUB1B protein levels are significantly higher in lung adenocarcinoma tissues compared to normal tissues, correlating with tumor stage, recurrence rate, and distant metastasis .

What is the expected molecular weight when detecting BUB1B by Western blot?

BUB1B appears at 120-130 kDa on Western blots, which aligns with its calculated molecular weight of 120 kDa (1050 amino acids) . When optimizing Western blot protocols:

• Use appropriate positive controls (K-562 cells, HeLa cells, human or mouse testis tissue)

• Start with antibody dilutions of 1:500-1:1000

• Be aware that post-translational modifications may cause slight variations in migration patterns

• Monitor for potential degradation products, especially in samples with high protease activity

The observed molecular weight (120-130 kDa) is consistent across multiple validated antibodies .

How should researchers design RNAi experiments targeting BUB1B to study its function in the spindle checkpoint?

Based on published methodologies, effective BUB1B knockdown experiments should:

• Test multiple siRNAs targeting different regions of BUB1B mRNA to identify the most efficient options

• Quantify knockdown efficiency by both:

  • Western blotting (aim for >25-fold depletion of protein)

  • Immunofluorescence at kinetochores (aim for 150-200 fold reduction)

• Use histone-H2B-GFP expressing cells for live-cell imaging to monitor chromosome movement

• Include proper controls (non-targeting siRNA)

• Measure key mitotic events:

  • Nuclear breakdown time

  • Chromosome congression time

  • Anaphase onset

Importantly, even low levels of kinetochore-localized BUB1B (2-5% of wild type) are sufficient to sustain a Mad2-dependent checkpoint response while still leading to chromosome misalignment . This underscores the importance of thorough validation of knockdown efficiency.

What controls and validations are essential when using BUB1B antibodies in cancer research studies?

For cancer research applications, rigorous experimental design should include:

• Expression controls:

  • Positive controls: Cell lines with known high BUB1B expression (HeLa, A-549, SK-LU-1)

  • Negative controls: Normal human bronchial epithelial cells (e.g., 16HBE)

  • Knockdown/knockout validation using siRNA or CRISPR/Cas9

• Technical validations:

  • Confirm specificity by showing absence of signal following BUB1B knockdown

  • Verify signal in multiple cell lines and tissue types

  • Use multiple antibodies targeting different epitopes when possible

• Functional validation:

  • Complement antibody detection with functional assays (proliferation, migration, invasion)

  • Correlate protein levels with clinical parameters and survival data

Studies have shown that knockdown of BUB1B results in significant inhibition of LUAD cell proliferation, invasion, and migration, which can be demonstrated through CCK-8 assays, colony formation, EDU/DAPI staining, transwell chambers, and wound-healing assays .

How can researchers accurately assess BUB1B localization dynamics during mitosis?

To accurately track BUB1B localization throughout mitosis:

• Imaging approaches:

  • Use 3D deconvolution microscopy for high-resolution imaging

  • Apply CREST antisera as a kinetochore reference marker (constant through cell cycle)

  • Implement live-cell imaging with fluorescently-tagged BUB1B for real-time dynamics

• Quantification methods:

  • Normalize BUB1B intensity to CREST signal for accurate comparisons between stages

  • Measure fluorescence intensity at individual kinetochores (≥10 kinetochores per cell)

  • Compare intensities across different mitotic phases (prometaphase vs. metaphase)

• Co-localization analysis:

  • Examine co-localization with other spindle checkpoint proteins (Mad1, Mad2, BubR1)

  • Assess relationship with microtubule attachment status using tubulin co-staining

Research has demonstrated that BUB1B localization patterns change dramatically depending on chromosome alignment and microtubule attachment status, with unaligned chromatids in BUB1B-depleted cells showing distinctive kinetochore morphology (flattened crescents spaced ~0.9 μm apart) .

How should researchers interpret contradictory BUB1B localization data across different studies?

Contradictory findings regarding BUB1B localization and function may result from:

• Experimental variables to consider:

  • Incomplete protein depletion (even 2-5% of wild-type BUB1B levels can sustain checkpoint function)

  • Cell type-specific differences in BUB1B regulation

  • Variations in fixation and staining protocols affecting epitope accessibility

  • Differences in mitotic synchronization methods

• Resolution strategies:

  • Carefully control for mitotic stage when comparing BUB1B localization

  • Quantify knockdown efficiency by both Western blot and immunofluorescence

  • Use multiple antibodies targeting different epitopes

  • Include direct comparison of experimental conditions from conflicting studies

For example, contradictory findings regarding Aurora B regulation of BUB1B localization have been reported. Some studies suggest Aurora B inhibition blocks kinetochore binding by BUB1B, while others show independent functioning. This discrepancy may be explained by variations in the extent of Aurora B inhibition and careful analysis of cell cycle stages .

What factors might contribute to variability in BUB1B antibody staining patterns in cancer tissues?

Variability in BUB1B immunostaining across cancer tissues may result from:

• Biological factors:

  • Tumor heterogeneity within and between patients

  • Cancer subtype-specific expression patterns

  • Genetic alterations affecting antibody epitopes

  • Stage-dependent changes in expression

• Technical factors:

  • Tissue fixation methods and duration

  • Antigen retrieval conditions (pH 9.0 TE buffer vs. pH 6.0 citrate buffer)

  • Different antibody clones recognizing distinct epitopes

  • Variations in detection systems (DAB vs. fluorescent)

Studies have shown that BUB1B expression correlates with tumor stage, recurrence rate, and distant metastasis in lung adenocarcinoma patients . Therefore, standardizing scoring methods and incorporating clinical data are essential for meaningful interpretation.

How can researchers reconcile discrepancies between mRNA and protein expression levels of BUB1B?

When encountering discrepancies between BUB1B mRNA and protein levels:

• Consider post-transcriptional regulation:

  • Assess microRNA regulation of BUB1B

  • Evaluate protein stability and half-life in your experimental system

  • Investigate post-translational modifications affecting protein stability

• Technical validation approaches:

  • Compare results across multiple antibodies and detection methods

  • Validate RNA-seq findings with RT-qPCR

  • Perform polysome profiling to assess translation efficiency

• Experimental strategies:

  • Combine techniques (e.g., RNA-FISH with IF) to analyze expression at single-cell level

  • Use protein degradation inhibitors to assess turnover rates

  • Implement pulse-chase experiments to measure protein half-life

Recent pan-cancer analyses have revealed consistent upregulation of BUB1B at both transcript and protein levels in most cancers , but individual samples may show discrepancies due to various regulatory mechanisms.

How does BUB1B antibody staining correlate with clinical outcomes in cancer patients?

BUB1B expression shows significant correlation with clinical parameters:

For optimal correlation with clinical outcomes:

• Use standardized scoring methods (H-score or percentage of positive cells)

• Stratify patients by BUB1B expression levels (high vs. low)

• Perform multivariate analysis adjusting for clinicopathological variables

• Correlate with key oncogenic pathways activation

What are the methodological considerations when using BUB1B antibodies to study its role in chemotherapy resistance?

When investigating BUB1B's role in chemotherapy resistance:

• Experimental design:

  • Generate stable BUB1B knockdown and overexpression cell lines

  • Test multiple chemotherapeutic agents at clinically relevant doses

  • Measure both short-term (viability) and long-term (clonogenic) responses

  • Evaluate BUB1B's relationship with known resistance mechanisms

• Mechanistic investigations:

  • Assess BUB1B complex formation with OTUD3 and NRF2

  • Evaluate downstream NRF2 signaling pathway activation

  • Measure ferroptosis sensitivity in BUB1B-modulated cells

  • Test combination treatment strategies (e.g., chemotherapy plus ML385)

Recent research has revealed that BUB1B forms a complex with OTUD3 and NRF2, stabilizing the NRF2 signaling pathway to facilitate insensitivity to ferroptosis and chemotherapy. In mouse models, a combined strategy of ML385 targeting and chemotherapy achieved synergistic effects in BUB1B-overexpressing tumors .

How should researchers design experiments to investigate BUB1B's interaction with other proteins using co-immunoprecipitation approaches?

For studying BUB1B protein interactions:

• Antibody selection for immunoprecipitation:

  • Choose antibodies with validated IP applications

  • Consider epitope location (N-terminal vs. C-terminal)

  • Test multiple antibodies to identify optimal performance

  • Validate specificity using BUB1B-depleted lysates

• Experimental conditions:

  • Optimize lysis buffers to preserve protein interactions

  • Consider cell synchronization to capture cell cycle-specific interactions

  • Include appropriate controls (IgG, input, knockout/knockdown samples)

  • Validate interactions using reciprocal co-IP approaches

• Detection strategies:

  • Use antibodies against endogenous proteins when possible

  • For weak or transient interactions, consider crosslinking approaches

  • Validate key interactions with proximity ligation assays

  • Complement co-IP with GST pulldown or yeast two-hybrid approaches

Studies have identified important interactions between BUB1B and ZNF143 in LUAD cells, as well as complex formation with OTUD3 and NRF2 affecting chemotherapy sensitivity . These findings highlight the importance of studying BUB1B's protein interaction network.

What are the methodological considerations when using BUB1B antibodies in single-cell analysis of tumor heterogeneity?

For single-cell applications of BUB1B antibodies:

• Technical optimization:

  • Validate antibody specificity at single-cell resolution

  • Optimize staining protocols for flow cytometry and CyTOF

  • Develop multiplexing strategies with other cell cycle markers

  • Establish quantitative imaging workflows for spatial context

• Analysis approaches:

  • Correlate BUB1B expression with cell cycle phases

  • Identify distinct cellular subpopulations based on BUB1B levels

  • Map BUB1B expression to tumor spatial architecture

  • Integrate with single-cell transcriptomics data

• Biological questions to address:

  • How does BUB1B expression vary within tumor regions?

  • Does BUB1B heterogeneity correlate with proliferative capacity?

  • Is BUB1B expression associated with particular cancer stem cell markers?

  • How does chromosome instability vary with BUB1B expression at single-cell level?

Single-cell analysis could help resolve conflicting findings regarding BUB1B's role in various cancers by revealing population heterogeneity not captured in bulk analyses.

How can researchers effectively combine BUB1B antibodies with other markers to study chromosome instability mechanisms?

To comprehensively study chromosome instability mechanisms:

• Multiplexed immunofluorescence approaches:

  • Combine BUB1B with kinetochore markers (CENP-A, Hec1)

  • Include spindle checkpoint proteins (Mad1, Mad2, Bub3)

  • Add chromosome segregation markers (Aurora B, CENP-E)

  • Incorporate DNA damage response proteins (γH2AX, 53BP1)

• Live-cell imaging strategies:

  • Use fluorescently-tagged BUB1B with H2B-RFP for chromosome tracking

  • Implement photoactivatable or photoswitchable fluorophores for dynamics

  • Apply FRET-based sensors to detect BUB1B activation states

  • Employ lattice light-sheet microscopy for high-resolution 4D imaging

• Functional readouts:

  • Measure micronuclei formation as CIN indicator

  • Quantify lagging chromosomes and anaphase bridges

  • Assess mitotic duration and checkpoint robustness

  • Analyze aneuploidy by metaphase spreads or single-cell sequencing

Research has shown that depletion of BUB1B results in distinct chromosome alignment defects, with unaligned chromatids showing abnormal microtubule attachments that are distinct from those observed with other checkpoint protein depletions .

What are the considerations when using BUB1B antibodies in combination with CRISPR/Cas9 genome editing approaches?

When combining BUB1B antibodies with CRISPR/Cas9 techniques:

• Experimental design:

  • Design guide RNAs targeting different BUB1B functional domains

  • Create both knockout and knock-in cell lines (e.g., fluorescent tags)

  • Generate domain-specific deletions to map antibody epitopes

  • Implement inducible CRISPR systems for temporal control

• Validation strategies:

  • Confirm editing efficiency by sequencing and protein expression

  • Verify antibody specificity using edited cell lines as controls

  • Assess off-target effects that might affect interpretation

  • Characterize phenotypic consequences of specific edits

• Advanced applications:

  • Use BUB1B antibodies to validate endogenous tagging efficiency

  • Create reporter cell lines for BUB1B dynamics studies

  • Generate complementation systems with BUB1B variants

  • Implement CRISPR screens for BUB1B interaction partners

A study investigating BUB1B's role in prostate cancer used CRISPR/Cas9 to generate clones with the recurrent BUB1B variant c.1171_1173del. Interestingly, they were unable to obtain heterozygous clones with monoallelic gene-editing for this variant, suggesting potential fitness constraints .