CDKB1-1 Antibody

Basic Research Questions

How to validate CDKB1-1 antibody specificity in plant tissue extracts?

• Methodological steps:

  • Use genetic controls (e.g., cdkb1;1 knockout mutants) to confirm absence of signal in Western blots .

  • Perform immunoprecipitation (IP) followed by mass spectrometry to identify co-purified proteins .

  • Compare antibody reactivity across plant species with known CDKB1-1 sequence divergence .

• Key data:

  Validation Method	Observation in Wild-Type	Observation in Mutant	Source
  Western Blot	~55 kDa band	No band
  IP-Kinase Assay	Activity peaks at 18h	No activity

What experimental controls are critical for CDKB1-1 localization studies?

• Include:

  • Tissue sections from cdkb1;1 mutants as negative controls .

  • Pre-immune serum to rule out nonspecific binding .

  • Cross-reactivity checks with CDKA (e.g., using antibodies against conserved epitopes) .

Advanced Research Questions

How to address contradictory CDKB1-1 expression patterns in germination vs. DNA damage responses?

• Analytical framework:

  • Distinguish cell-cycle-phase-specific roles (e.g., G2/M vs. S-phase) .

  • Quantify post-translational modifications (e.g., phosphorylation at Thr-161) under stress .

  • Correlate expression with CYCB1 partners using co-IP .

• Example: CDKB1-1 binds D-type cyclins during germination but associates with CYCB1 after cisplatin-induced DNA damage .

What strategies resolve CDKB1-1 cross-reactivity with CDKA in kinase assays?

• Solutions:

  • Use isoform-specific antibodies (e.g., anti-CDKB1;1 N-terminal) .

  • Employ sequential IP: First remove CDKA complexes using anti-CDKA beads, then isolate CDKB1-1 .

  • Validate using cdka;1 mutants as additional controls .

How to design co-immunoprecipitation experiments for CDKB1-1–CYCB1 complexes?

• Protocol:

  • Extract proteins with non-denaturing lysis buffer (e.g., 50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40) .

  • Pre-clear lysates with protein A/G beads before adding anti-CDKB1;1 antibody .

  • Verify interactions via in vitro kinase assays using histone H1 or RAD51 as substrates .

What functional assays confirm CDKB1-1’s role in homologous recombination (HR)?

• Approaches:

  • Measure RAD51 focus formation in cdkb1;1 mutants after cisplatin treatment .

  • Quantify HR efficiency using reporter lines (e.g., GFP-based recombination assays) .

  • Test phosphorylation of RAD51 by CDKB1-1–CYCB1 complexes in vitro .

• Data:

  Assay	Wild-Type Result	cdkb1;1 Mutant Result	Source
  RAD51 Foci	12–15 foci/nucleus	≤3 foci/nucleus
  HR Efficiency	85% repair success	22% repair success

Methodological Best Practices

• Antibody Storage: Aliquot and store at −80°C in glycerol-based buffer to prevent freeze-thaw degradation.

• Quantitative Analysis: Normalize CDKB1-1 signal to housekeeping proteins (e.g., actin) only in proliferating tissues .

• Conflict Resolution: If kinase activity contradicts mRNA levels, check proteasomal degradation via MG132 treatment .