BUB3.1 Antibody

Basic Research Questions

• How can I validate the specificity of a BUB3 antibody in mammalian cell lines?

  • Method: Use siRNA knockdown or CRISPR-Cas9-generated BUB3 knockout cells. Compare antibody signals in treated vs. untreated samples via Western blot (WB) or immunofluorescence (IF). For example, silencing BUB3 with siRNA oligos (e.g., Dharmacon duplex: SEQ ID NO. 4–5) reduces signal intensity by >70% in WB .

  • Controls: Include a non-targeting siRNA and validate knockdown efficiency with qPCR. For WB, use lysates from cells with confirmed BUB3 deletion .

• What are the recommended applications for BUB3 antibodies in human tissue studies?

  • Applications:

    Application	Dilution Range	Validation Example
    Western Blot	1:500–1:4,000	Detects 37–40 kDa band in HEK-293T/MCF-7 lysates
    IHC (FFPE)	1:50–1:200	Cytoplasmic/nuclear staining in pancreatic cancer
    Immunoprecipitation	1:10–1:50	Co-IP with Bub1 in NIH3T3/T cells

• How do I troubleshoot cross-reactivity between BUB3 and Bub1 antibodies?

  • Approach:

    • Use antibodies validated for <1% cross-reactivity (e.g., Human/Mouse Bub3 Antibody AF5706) .

    • Preclear lysates with protein A/G beads to reduce nonspecific binding.

    • For WB, compare migration patterns: Bub1 (~120 kDa) vs. BUB3 (~37 kDa) .

Advanced Research Questions

• How do Bub3-Bub1 interactions influence spindle checkpoint signaling in transformed cells?

  • Experimental Design:

    • Use co-immunoprecipitation (co-IP) in SV40 T antigen-expressing cells (e.g., SV4 rat fibroblasts). T antigen enhances Bub1 kinase activity but does not bind Bub3 directly .

    • Key Finding: Bub3 co-IPs with Bub1 only when T antigen is present at permissive temperatures (33°C), suggesting temperature-dependent complex stability .

• What experimental controls are critical when studying BUB3 in aneuploidy models?

  • Controls:

    Control Type	Purpose	Example
    Irrelevant antibody IP	Rule out nonspecific binding	Mouse IgG in NIH3T3/T lysates
    BUB3 siRNA rescue	Confirm phenotype specificity	Restore WT BUB3 in knockdown cells
    Mitotic arrest agents	Validate checkpoint activation	Treat with nocodazole/oryzalin

• How can conflicting localization data for BUB3 (nuclear vs. kinetochore) be resolved?

  • Methodology:

    • Synchronize cells at prometaphase (e.g., thymidine block + release).

    • Use dual IF with kinetochore markers (e.g., CREST antisera). In Arabidopsis, BUB3.3-BMF3 colocalizes at kinetochores only during checkpoint activation .

    • Note: Cytoplasmic BUB3 correlates with cancer recurrence; validate subcellular localization contextually .

• What structural motifs in BUB3 are critical for its interaction with Bub1?

  • Key Findings:

    • Bub1’s N-terminal (aa 1–303) and C-terminal (aa 691–1085) regions bind Bub3 .

    • In Arabidopsis, BUB3.3 binds BMF3 via two internal repeat motifs (IR1/IR2); IR2 is essential for SAC function .

    • Implication: Truncate BUB3 at IR1/IR2 to study binding thermodynamics (e.g., yeast two-hybrid assays) .

Data Contradiction Analysis

• Why do some studies report BUB3 as dispensable for immortalization but essential for transformation?

  • Resolution:

    • SV40 T antigen mutants (e.g., dl89–97) bind Bub1 but not Bub3. These mutants immortalize cells but fail to override the spindle checkpoint, linking Bub3 to transformation-specific pathways .

    • Experimental Tip: Compare BUB3 knockout vs. WT cells in soft-agar assays to isolate transformation roles .

Species-Specific Considerations

• How does BUB3 antibody reactivity vary between human, mouse, and plant models?

  • Cross-Reactivity Data:

    Antibody	Species Reactivity	Epitope
    ab264121 (Abcam)	Human	aa 1–50 (N-terminal)
    A03118-1 (Boster)	Human, Mouse, Rat	Recombinant N-terminus
    AF5706 (R&D Systems)	Human, Mouse	WD40 repeats

  • Recommendation: For plant studies (e.g., Arabidopsis BUB3.1/3.2), use isoform-specific antibodies due to low sequence homology with mammalian BUB3 .

Methodological Optimization

• How to optimize BUB3 detection in low-abundance samples (e.g., primary cells)?

  • Strategy:

    • Use signal amplification (e.g., tyramide-based IHC) .

    • Precipitate proteins with TCA/acetone to concentrate lysates.

    • For WB, load ≥50 µg lysate and use high-sensitivity substrates (e.g., ECL Prime) .