NBS1 Antibody

Here’s a structured collection of research-focused FAQs for NBS1 antibodies, designed to address experimental design, data interpretation, and methodological challenges in academic settings:

How do I validate NBS1 antibody specificity for Western blot?

• Methodology:

  • Use knockout (KO) cell lines (e.g., HeLa NBS1 KO) as negative controls. Probe lysates from parental and KO cells side-by-side.

  • Validate with post-translational modification (PTM)-insensitive antibodies if studying phosphorylated isoforms.

  • Compare observed band sizes with expected molecular weights (e.g., 95 kDa for full-length NBS1 , 84 kDa for cleaved forms ).

Example Validation Table:

What experimental factors affect NBS1 detection in immunofluorescence?

• Critical Parameters:

  • Fixation: Methanol fixation outperforms paraformaldehyde for nuclear localization studies .

  • Permeabilization: 0.1% Triton X-100 for 10 min preserves epitope accessibility .

  • Antibody Dilution: Optimize between 1:50 (low-abundance targets) and 1:200 (high-expression systems) .

How to resolve discrepancies in reported NBS1 molecular weights across studies?

• Analysis Framework:

  • Species Differences: Human NBS1 migrates at ~95 kDa , while murine orthologs may appear lighter .

  • PTMs: Phosphorylation (e.g., Ser343 after DNA damage) increases apparent molecular weight .

  • Proteolytic Cleavage: Caspase-mediated cleavage during apoptosis generates ~70 kDa fragments .

Molecular Weight Discrepancy Table:

How to optimize co-immunoprecipitation (Co-IP) of the MRN complex?

• Protocol Adjustments:

  • Crosslinking: Use DSS (disuccinimidyl suberate) to stabilize transient interactions .

  • Nuclease Treatment: Benzonase eliminates DNA-mediated artifactual binding .

  • Buffer Composition: TBS-T + 150 mM NaCl reduces non-specific binding .

Co-IP Optimization Checklist:

• ✔️ Pre-clear lysates with Protein A/G beads

• ✔️ Include IgG isotype controls

• ✔️ Validate with reciprocal IP (e.g., anti-MRE11 after anti-NBS1 pull-down)

How to integrate NBS1 studies with ATM/ATR pathway analysis?

• Multiplex Approaches:

  • Sequential Blotting: Re-probe Western blot membranes with anti-phospho-ATM (Ser1981) after NBS1 detection .

  • Comet-FISH: Combine neutral comet assays (for DSBs) with NBS1 immunofluorescence to link localization to damage severity .

  • Checkpoint Activation: Use thymidine block-and-release assays with NBS1 siRNA to quantify S-phase arrest defects .

Why does NBS1 antibody staining appear cytoplasmic in some cancer models?

• Troubleshooting Guide:

  • Artifact Check: Confirm nuclear-cytoplasmic fractionation efficiency via Lamin B1/GAPDH controls .

  • Biological Relevance: Pathogenic NBS1 mutants (e.g., Δ657-658) mislocalize due to disrupted nuclear localization signals .

  • Fixation Artifacts: Over-fixation with methanol can collapse nuclear structure; test alternative protocols .

How to reconcile contradictory roles of NBS1 in homologous recombination (HR) vs. non-homologous end joining (NHEJ)?

• Experimental Design:

  • Pathway-Specific Assays:

    • HR efficiency: DR-GFP reporter + NBS1 knockdown .

    • NHEJ efficiency: EJ5-GFP reporter + γH2AX co-staining .

  • Context Dependency: NBS1 promotes HR in S/G2 phase but stabilizes NHEJ factors (e.g., Ku70/80) in G1 .

Technical Notes

• Antibody Storage: Unpurified polyclonals (e.g., NB100-143) degrade rapidly; aliquot and avoid freeze-thaw cycles .

• Multiplexing: Pair rabbit anti-NBS1 with mouse anti-γH2AX for dual IF, using cross-adsorbed secondaries .