BUBR1 Antibody

What is BubR1 and why is it important in cell division research?

BubR1 (also known as BUB1B) is an essential component of the mitotic checkpoint complex with an approximate molecular weight of 120-130 kDa. It plays a crucial role in the spindle assembly checkpoint (SAC) that delays anaphase until all chromosomes are properly attached to the mitotic spindle . BubR1 is vital for:

• Monitoring kinetochore-microtubule attachment during mitosis

• Inhibiting the activity of the anaphase-promoting complex/cyclosome (APC/C)

• Preventing chromosome missegregation and aneuploidy

• Maintaining genomic stability
  BubR1 dysfunction has been associated with chromosome instability in colorectal cancers and its expression levels have been linked to survival outcomes in cholangiocarcinoma , making it an important target for both basic cell biology and cancer research.

What are the key applications for BubR1 antibodies in mitosis research?

BubR1 antibodies are versatile tools for studying mitotic checkpoint mechanisms with several validated applications:

How can I optimize Western blotting conditions for detecting BubR1?

Optimizing Western blot conditions for BubR1 detection requires careful consideration of several factors:

• Sample preparation:

  • For mitotic cells, nocodazole treatment (typically 100 ng/ml) can enrich for cells with active BubR1

  • Use mitotic shake-off to collect mitotic cells with high BubR1 expression

  • Include protease inhibitors in lysis buffer to prevent degradation

• Gel electrophoresis:

  • Use Tris-HCl polyacrylamide gels to properly resolve the 120-130 kDa BubR1 protein

  • Load appropriate positive controls (e.g., HeLa cells, K-562 cells, or testis tissue)

• Antibody selection and dilution:

  • Start with recommended dilution (1:500-1:1000) and optimize if needed

  • Consider using antibodies that recognize specific regions or modifications of BubR1, such as acetylated BubR1 (K250)

• Detection methods:

  • Enhanced chemiluminescence (ECL) systems work well for BubR1 detection

  • For quantification, use 16-bit TIFF format images and analysis with ImageJ software
    For comparative analyses, use β-tubulin or similar housekeeping proteins as loading controls, and include both interphase and M-phase samples to observe cell cycle-dependent changes in BubR1 levels and modifications .

What controls should be included when studying BubR1 localization by immunofluorescence?

Robust immunofluorescence studies of BubR1 require careful experimental design and appropriate controls:

• Positive controls:

  • Include cells in prometaphase with unattached kinetochores (e.g., nocodazole-treated cells), which should show strong BubR1 kinetochore localization

  • Use known BubR1-positive cell lines such as HeLa or SKOV-3 cells

• Negative controls:

  • Cells in anaphase (when BubR1 normally dissociates from kinetochores)

  • Secondary antibody-only controls to assess background fluorescence

  • BubR1-depleted cells (siRNA-treated) to confirm antibody specificity

• Co-staining markers:

  • Centromere/kinetochore markers (e.g., CENP-E, anticentromere antibodies) to confirm BubR1 localization

  • Microtubule staining (anti-α-tubulin) to assess kinetochore-microtubule attachment status

  • DNA counterstaining (DAPI) to identify mitotic stages

• Technical considerations:

  • Optimize fixation and permeabilization protocols (paraformaldehyde fixation followed by detergent permeabilization works well)

  • Use appropriate blocking solutions to minimize non-specific binding

  • Acquire images with proper exposure settings to avoid saturation
    When analyzing BubR1 kinetochore localization, compare cells at different mitotic stages and under different treatment conditions (e.g., before and after spindle poisons or Aurora B inhibitors) to observe dynamic changes in localization patterns .

How do post-translational modifications affect BubR1 function during mitosis?

BubR1 undergoes several post-translational modifications that critically regulate its function during mitosis:

• Acetylation:

  • BubR1 is acetylated at lysine 250 (K250) specifically during M phase

  • Acetylation can be detected using specific anti-Ac-K250 antibodies

  • This modification is important for modulating APC/C activity and BubR1 function in the SAC

• Phosphorylation:

  • BubR1 is phosphorylated by multiple kinases during mitosis

  • Phosphorylation states affect its interaction with other checkpoint proteins and its kinase activity

  • Different phosphorylation sites have distinct roles in checkpoint signaling

• Ubiquitination and degradation:

  • BubR1 is continuously synthesized and degraded during mitosis

  • Treatment with cycloheximide (CHX) leads to decreased BubR1 levels, indicating ongoing protein turnover

  • The balance between synthesis and degradation is important for maintaining appropriate BubR1 levels
    To study these modifications experimentally:

• Use phospho-specific or acetyl-specific antibodies for detection of modified forms

• Employ immunoprecipitation followed by Western blotting with modification-specific antibodies

• Compare modifications under different conditions (e.g., nocodazole arrest versus normal mitotic progression)

• Consider using inhibitors of specific modifying enzymes to assess functional consequences
  Understanding these modifications is critical for unraveling the complex regulation of BubR1 during the cell cycle and its role in preventing chromosome missegregation.

What are the functional differences between BubR1 and Bub1, and how can they be distinguished experimentally?

BubR1 and Bub1 originated from duplication of an ancestor gene but have evolved distinct functions in the spindle assembly checkpoint:

• Antibody specificity:

  • Use validated antibodies that specifically recognize either BubR1 or Bub1

  • Confirm specificity by immunoprecipitation experiments (anti-Bub1 immunoprecipitates should not be recognized by anti-BubR1 antibodies, and vice versa)

• Functional assays:

  • BubR1 depletion specifically affects SAC sustainability and MCC formation

  • Bub1 depletion affects initial kinetochore recruitment of checkpoint proteins

  • Different mutant constructs (e.g., BubR1 with Bub1 loop regions) can be used to assess specific domain functions

• Localization studies:

  • During early mitosis, both proteins localize to kinetochores but with different dynamics

  • Co-immunostaining can reveal their relative localization patterns
    Understanding these differences is critical when designing experiments targeting specific aspects of the spindle assembly checkpoint pathway.

How can I assess BubR1's role in spindle assembly checkpoint function?

Several experimental approaches can effectively evaluate BubR1's function in the spindle assembly checkpoint:

• Nocodazole challenge assay:

  • Treat cells with nocodazole (typically 100 ng/ml) to depolymerize microtubules and activate the SAC

  • Measure duration of mitotic arrest by time-lapse imaging of cells expressing fluorescent markers (e.g., H2B-RFP)

  • Compare wild-type cells with BubR1-depleted or BubR1-overexpressing cells

  • BubR1-depleted cells show shortened mitotic arrest, while certain BubR1 overexpression can extend arrest duration

• Mps1 inhibition assay:

  • Use Mps1 inhibitors (e.g., AZ3146) at different concentrations to weaken SAC signaling

  • Monitor mitotic timing in the presence of both nocodazole and Mps1 inhibitor

  • BubR1 overexpression can enhance checkpoint sustainability even under partial Mps1 inhibition

• Monastrol washout assay:

  • Treat cells with monastrol to create monopolar spindles, then wash out to allow error correction

  • Assess correction of attachment errors with or without Aurora B inhibition

  • BubR1 overexpression improves error correction capacity

• Microinjection of anti-BubR1 antibodies:

  • Microinject anti-BubR1 antibodies into living cells

  • Monitor effects on mitotic progression and checkpoint function

  • BubR1 antibody injection abolishes nocodazole-induced mitotic arrest

• siRNA-mediated knockdown followed by rescue experiments:

  • Deplete endogenous BubR1 using siRNA

  • Express siRNA-resistant wild-type or mutant BubR1 constructs

  • Assess rescue of checkpoint function and chromosome segregation
    These experimental approaches provide complementary information about BubR1's multifaceted roles in checkpoint signaling and chromosome segregation.

What experimental approaches can reveal BubR1's interactions with other mitotic proteins?

Understanding BubR1's protein interactions is crucial for elucidating its role in mitotic checkpoint function:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate BubR1 from mitotic cell extracts using specific antibodies

  • Analyze co-precipitating proteins by Western blot or mass spectrometry

  • Compare interactomes between different mitotic stages or treatments

  • Key BubR1 interactors include CDC20, Mad2, Bub3, APC/C subunits, and CENP-E

• Domain deletion and mutation analysis:

  • Generate BubR1 constructs with specific domain deletions or mutations:

    • BubR1 ΔN (lacks N-terminal domain)

    • BubR1 ΔI (internal deletion)

    • BubR1 ΔPhe (deletion of Phe box)

    • BubR1 ΔD (deletion of D-box2)

    • BubR1 ΔKARD (deletion of kinetochore attachment regulatory domain)

  • Express these constructs in cells and assess their ability to:

    • Localize to kinetochores

    • Interact with checkpoint proteins

    • Rescue checkpoint function in BubR1-depleted cells

• Proximity ligation assays:

  • Detect in situ protein-protein interactions between BubR1 and its binding partners

  • Visualize interactions at specific subcellular locations (e.g., kinetochores)

• Yeast two-hybrid or mammalian two-hybrid assays:

  • Identify direct protein-protein interactions

  • Map interaction domains between BubR1 and binding partners

• In vitro binding assays with recombinant proteins:

  • Express and purify recombinant BubR1 domains

  • Test direct binding to purified interaction partners

  • Determine binding affinities and kinetics
    These approaches have revealed that BubR1 forms multiple protein complexes during mitosis, including:

• The mitotic checkpoint complex (MCC) with CDC20, Mad2, and Bub3

• Kinetochore-associated complexes with CENP-E

• Interactions with the APC/C

• Heterodimeric complexes with Bub1
  Understanding these interaction networks is essential for developing targeted approaches to modulate BubR1 function in research and potential therapeutic applications.

How should I address low signal issues when detecting BubR1 in Western blots?

When encountering weak BubR1 signals in Western blots, consider these methodological solutions:

• Sample preparation optimization:

  • Enrich for mitotic cells using nocodazole treatment (100 ng/ml) to increase BubR1 abundance

  • Use mitotic shake-off to collect cells with high BubR1 expression

  • Add proteasome inhibitors (e.g., MG132) to prevent BubR1 degradation

  • Remember that BubR1 is continuously synthesized and degraded during mitosis

• Antibody selection and optimization:

  • Test different antibodies that recognize different epitopes of BubR1

  • Optimize antibody concentration beyond recommended range (typically 1:500-1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try different blocking agents (BSA vs. non-fat dry milk)

• Detection system enhancement:

  • Use high-sensitivity ECL substrates for horseradish peroxidase detection

  • Consider signal amplification systems

  • For quantitative Western blots, use 16-bit TIFF format images for analysis

• Loading control considerations:

  • Ensure equal loading with housekeeping proteins (tubulin, GAPDH)

  • Compare your samples to known positive controls (HeLa cells, K-562 cells, testis tissue)

• Technical improvements:

  • Use fresh transfer buffers and optimize transfer conditions

  • Consider gradient gels for better resolution of the 120-130 kDa BubR1 protein

  • Reduce washing stringency if signal is weak
    If BubR1 signal remains problematic despite these optimizations, consider alternative approaches such as immunoprecipitation followed by Western blotting to concentrate the protein before detection.

What are common issues with BubR1 immunofluorescence staining and how can they be resolved?

Achieving optimal BubR1 immunofluorescence staining requires addressing several common challenges:

• Weak kinetochore signals:

  • Problem: BubR1 signals at kinetochores appear weak or undetectable

  • Solutions:

    • Optimize fixation method (paraformaldehyde concentration and time)

    • Try different permeabilization agents (Triton X-100, digitonin, or methanol)

    • Use antigen retrieval techniques if necessary

    • Increase antibody concentration or incubation time

    • Enrich for prometaphase cells using nocodazole treatment

    • Use signal amplification systems

• High background staining:

  • Problem: Non-specific staining obscures specific BubR1 signals

  • Solutions:

    • Optimize blocking conditions (try different blocking agents and times)

    • Increase washing duration and stringency

    • Pre-absorb antibodies with cell extracts

    • Titrate primary and secondary antibodies to optimal concentrations

    • Use highly cross-adsorbed secondary antibodies

• Inconsistent staining patterns:

  • Problem: Variable BubR1 staining between experiments or cells

  • Solutions:

    • Standardize fixation timing (BubR1 localization changes during mitotic progression)

    • Ensure consistent antibody handling and storage

    • Include positive controls in every experiment

    • Use rat anti-BubR1 antibodies for detection when rabbit antibodies are used for injection experiments

• Co-staining challenges:

  • Problem: Difficulty visualizing BubR1 along with other kinetochore proteins

  • Solutions:

    • Carefully select compatible primary antibodies from different host species

    • Use zenon labeling or directly conjugated antibodies

    • Apply sequential staining protocols for challenging combinations

    • Consider fluorescent protein tagging for live cell imaging

• Image acquisition settings:

  • Problem: BubR1 signals appear different across microscopes or sessions

  • Solutions:

    • Standardize exposure settings based on positive controls

    • Use appropriate dynamic range for image acquisition

    • Apply consistent post-acquisition processing
      Including appropriate controls (antibody specificity, siRNA-depleted cells, different mitotic stages) will help validate the observed staining patterns and troubleshoot potential issues.

How can BubR1 antibodies be utilized to study chromosomal instability in cancer?

BubR1 antibodies offer powerful tools for investigating chromosomal instability (CIN) in cancer through multiple experimental approaches:

• Expression analysis in tumor samples:

  • Use immunohistochemistry with anti-BubR1 antibodies to assess expression levels in cancer tissues

  • Apply H-score methodology for quantification: H-score = [1 × (% of 1+ cells)] + [2 × (% of 2+ cells)] + [3 × (% of 3+ cells)]

  • Compare BubR1 expression between tumor and adjacent normal tissues

  • Correlate expression with clinical parameters (survival, stage, grade)

  • In cholangiocarcinoma, high BubR1 expression correlates with poor survival

• Functional studies in cancer cell lines:

  • Modulate BubR1 levels using siRNA knockdown or overexpression

  • Assess effects on:

    • Cell viability and proliferation

    • Colony formation capacity

    • Cell cycle distribution (using flow cytometry)

    • Migration and invasion potential

    • Response to chemotherapeutic agents

• Chromosomal instability assessment:

  • Combine BubR1 antibody staining with FISH or immunofluorescence for centromere markers

  • Quantify chromosome missegregation events in anaphase cells

  • Measure aneuploidy through karyotype analysis or DNA content assessment

  • Evaluate micronuclei formation as a marker of chromosome missegregation

• Mutations and alterations analysis:

  • Identify BubR1 mutations in colorectal cancers with chromosome instability

  • Express mutant BubR1 constructs and assess their effects on mitotic checkpoint function

  • Evaluate how cancer-associated BubR1 mutations affect protein interactions and localization
    These approaches have revealed that BubR1 alterations contribute to cancer development through effects on genomic stability, with both reduced and increased expression potentially playing roles in different cancer contexts.

What methodologies are most effective for studying BubR1's role in cancer therapeutic resistance?

Investigating BubR1's contribution to therapeutic resistance requires specialized experimental approaches:

• Cell line models of acquired resistance:

  • Develop resistant cancer cell lines through continuous drug exposure

  • Compare BubR1 expression, localization, and post-translational modifications between parental and resistant lines using Western blot and immunofluorescence

  • Assess chromosome segregation fidelity in resistant versus sensitive cells

• Manipulation of BubR1 levels and activity:

  • Use siRNA to knockdown BubR1 in resistant cells and test re-sensitization

  • Overexpress wild-type or mutant BubR1 in sensitive cells to induce resistance

  • Apply the Combination Index analysis to evaluate synergistic effects of BubR1 targeting with conventional therapies

• Checkpoint adaptation studies:

  • Use BubR1 antibodies to monitor checkpoint activation and silencing in response to therapy

  • Track mitotic duration and fate after treatment using time-lapse microscopy

  • Compare checkpoint sustainability between sensitive and resistant cells using nocodazole challenge assays

• BubR1 interaction with oncogenic pathways:

  • Study effects of oncogene activation (e.g., KRas G12V) on BubR1 function

  • Assess how therapeutic targeting of oncogenic pathways affects BubR1 activity

  • Investigate downstream effects of BubR1 alterations on apoptotic pathways

• Clinical correlation studies:

  • Analyze BubR1 expression in pre- and post-treatment patient samples

  • Correlate BubR1 levels with treatment response and progression-free survival

  • Develop predictive biomarkers based on BubR1 status
    These methodologies have revealed that manipulation of BubR1 levels can alter cancer cell sensitivity to therapeutic agents and influence cell survival mechanisms. For instance, BubR1 knockdown in cholangiocarcinoma cell lines affects their viability, migration, and invasion capabilities, suggesting potential therapeutic implications .